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Understanding of microbial communities inhabiting cattle vaginal tract may lead to a better comprehension of bovine physiology and reproductive health being of great economic interest. Up to date, studies involving cattle microbiota are focused on the gastrointestinal tract, and little is known about the vaginal microbiota. This study aimed to investigate the vaginal microbiome in Nellore cattle, heifers and cows, pregnant and non-pregnant, using a culture independent approach. The main bacterial phyla found were Firmicutes (~40-50%), Bacteroidetes (~15-25%) and Proteobacteria (~5-25%), in addition to ~10-20% of non-classified bacteria. 45-55% of the samples were represented by only ten OTUs: Aeribacillus, Bacteroides, Clostridium, Ruminococcus, Rikenella, Alistipes, Bacillus, Eubacterium, Prevotella and non-classified bacteria. Interestingly, microbiota from all 20 animals could be grouped according to the respiratory metabolism of the main OTUs found, creating three groups of vaginal microbiota in cattle. Archaeal samples were dominated by the Methanobrevibacter genus (Euryarchaeota, ~55-70%). Ascomycota was the main fungal phylum (~80-95%) and Mycosphaerella the most abundant genus (~70-85%). Hormonal influence was not clear, but a tendency for the reduction of bacterial and increase of archaeal populations in pregnant animals was observed. Eukaryotes did not vary significantly between pregnant and non-pregnant animals, but tended to be more abundant on cows than on heifers. The present work describes a great microbial variability in the vaginal community among the evaluated animals and groups (heifers and cows, pregnant and non-pregnant), which is significantly different from the findings previously reported using culture dependent methods, pointing out the need for further studies on this issue. The microbiome found also indicates that the vaginal colonization appears to be influenced by the gastrointestinal community.

Once inside a vertebrate host after infection, individual schistosomula of the parasite Schistosoma mansoni find a new and complex environment, which requires quick adjustments for survival, such as those that allow it to avoid the innate immune response of the host. Thus, it is very important for the parasite to remain within the skin after entering the host for a period of about 3 days, at which time it can then reach the venous system, migrate to the lungs and, by the end of eighth day post-infection, it reach the portal venous system, while undergoing minimal changes in morphology. However, after just a few days in the portal blood system, the parasite experiences an extraordinary increase in biomass and significant morphological alterations. Therefore, determining the constituents of the portal venous system that may trigger these changes that causes the parasite to consolidate its development inside the vertebrate host, thus causing the disease schistosomiasis, is essential. The present work simulated the conditions found in the portal venous system of the vertebrate host by exposing schistosomula of S. mansoni to in vitro culture in the presence of portal serum of the hamster, Mesocricetus auratus. Two different incubation periods were evaluated, one of 3 hours and one of 12 hours. These time periods were used to mimic the early contact of the parasite with portal serum during the course of natural infection. As a control, parasites were incubated in presence of hamster peripheral serum, in order to compare gene expression signatures between the two conditions. The mRNA obtained from parasites cultured under both conditions were submitted to a whole transcriptome library preparation and sequenced with a next generation platform. On average, nearly 15 million reads were produced per sample and, for the purpose of gene expression quantification, only reads mapped to one location of the transcriptome were considered. After statistical analysis, we found 103 genes differentially expressed by schistosomula cultured for 3 hours and 12 hours in the presence of hamster portal serum. After the subtraction of a second list of genes, also differentially expressed between schistosomula cultured for 3 hours and 12 hours in presence of peripheral serum, a set of 58 genes was finally established. This pattern was further validated for a subset of 17 genes, by measuring gene expression through quantitative real time polymerase chain reaction (qPCR). Processes that were activated by the portal serum stimulus include response to stress, membrane transport, protein synthesis and folding/degradation, signaling, cytoskeleton arrangement, cell adhesion and nucleotide synthesis. Additionally, a smaller number of genes down-regulated under the same condition act on cholinergic signaling, inorganic cation and organic anion membrane transport, cell adhesion and cytoskeleton arrangement. Considering the role of these genes in triggering processes that allow the parasite to quickly adapt, escape the immune response of the host and start maturation into an adult worm after contact with the portal serum, this work may point to unexplored molecular targets for drug discovery and vaccine development against schistosomiasis.

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

Introduction Zika virus (ZIKV) caused an outbreak in Brazil, in 2015, being associated to microcephaly. ZIKV has a strong neurotropism leading to death of infected cells in different brain regions, including the hippocampus, a major site for neurogenesis. The neuronal populations of the brain are affected differently by ZIKV from Asian and African ancestral lineages. However, it remains to be investigated whether subtle variations in the ZIKV genome can impact hippocampus infection dynamics and host response. Objective This study evaluated how two Brazilian ZIKV isolates, PE243 and SPH2015, that differ in two specific missense amino acid substitutions, one in the NS1 protein and the other in the NS4A protein, affect the hippocampal phenotype and transcriptome. Methods Organotypic hippocampal cultures (OHC) from infant Wistar rats were infected with PE243 or SPH2015 and analyzed in time series using immunofluorescence, confocal microscopy, RNA-Seq and RT-qPCR. Results Unique patterns of infection and changes in neuronal density in the OHC were observed for PE243 and SPH2015 between 8 and 48 h post infection (p.i.). Phenotypic analysis of microglia indicated that SPH2015 has a greater capacity for immune evasion. Transcriptome analysis of OHC at 16 h p.i. disclosed 32 and 113 differentially expressed genes (DEGs) in response to infection with PE243 and SPH2015, respectively. Functional enrichment analysis suggested that infection with SPH2015 activates mostly astrocytes rather than microglia. PE243 downregulated biological process of proliferation of brain cells and upregulated those associated with neuron death, while SPH2015 downregulated processes related to neuronal development. Both isolates downregulated cognitive and behavioral development processes. Ten genes were similarly regulated by both isolates. They are putative biomarkers of early hippocampus response to ZIKV infection. At 5, 7, and 10 days p.i., neuronal density of infected OHC remained below controls, and mature neurons of infected OHC showed an increase in the epigenetic mark H3K4me3, which is associated to a transcriptionally active state. This feature is more prominent in response to SPH2015. Conclusion Subtle genetic diversity of the ZIKV affects the dynamics of viral dissemination in the hippocampus and host response in the early stages of infection, which may lead to different long-term effects in neuronal population.

The interplay between bacterial virulence factors and the host innate immune response in pneumococcal meningitis (PM) can result in uncontrolled neuroinflammation, which is known to induce apoptotic death of progenitor cells and post-mitotic neurons in the hippocampal dentate gyrus, resulting in cognitive impairment. Vitamin B12 attenuates hippocampal damage and reduces the expression of some key inflammatory genes in PM, by acting as an epidrug that promotes DNA methylation, with increased production of S-adenosyl-methionine, the universal donor of methyl. Eleven-day-old rats were infected with via intracisternal injection and then administered either vitamin B12 or a placebo. After 24 hours of infection, the animals were euthanized, and apoptosis in the hippocampal dentate gyrus, microglia activation, and the inflammatory infiltrate were quantified in one brain hemisphere. The other hemisphere was used for RNA-Seq and RT-qPCR analysis. In this study, adjuvant therapy with B12 was found to modulate the hippocampal transcriptional signature induced by PM in infant rats, mitigating the effects of the disease in canonical pathways related to the recognition of pathogens by immune cells, signaling via NF-kB, production of pro-inflammatory cytokines, migration of peripheral leukocytes into the central nervous system, and production of reactive species. Phenotypic analysis revealed that B12 effectively inhibited microglia activation in the hippocampus and reduced the inflammatory infiltrate in the central nervous system of the infected animals. These pleiotropic transcriptional effects of B12 that lead to neuroprotection are partly regulated by alterations in histone methylation markings. No adverse effects of B12 were predicted or observed, reinforcing the well-established safety profile of this epidrug. B12 effectively mitigates the impact of PM on pivotal neuroinflammatory pathways. This leads to reduced microglia activation and inflammatory infiltrate within the central nervous system, resulting in the attenuation of hippocampal damage. The anti-inflammatory and neuroprotective effects of B12 involve the modulation of histone markings in hippocampal neural cells.

Describing the bovine vaginal microbiota is essential to better understand its physiology and its impact on health maintenance. Despite the economic importance of reproduction of these animals, bovine vaginal microbial community is still poorly described in comparison with rumen microbiome. Previous studies of our group described the vaginal microbiota of Nellore, an important Bos taurus indicus breed, using metagenomics. In order to better understand this microbiota, the present work aims to investigate another important breed, Gyr. Results have shown bacterial dominance over Archaea and Fungi was observed, with the most abundant bacterial phylum (Firmicutes) representing 40–50% of bacterial population, followed by Bacteroidetes, Proteobacteria, and Actinobacteria. The Fungi kingdom had the Mycosphaerella genus as its main representative, followed by Cladosporium. Archaea were observed at a very low abundance in all animals, with a high relative abundance of Methanobrevibacter genus. These results demonstrate a high microbial diversity on vaginal tract of Gyr, as demonstrated for Nellore and different from the previously described for other species. Our results indicate a great similarity between vaginal microbiota of Nellore and Gyr despite the differences in animal handling and genetic improvement. As observed for both breeds, individual variation is the largest source of microbial diversity between animals.

Whole genome sequencing of bovine breeds has allowed identification of genetic variants in milk protein genes. However, functional repercussion of such variants at a molecular level has seldom been investigated. Here, the results of a multistep Bioinformatic analysis for functional characterization of recently identified genetic variants in Brazilian Gyr and Guzerat breeds is described, including predicted effects on the following: (i) evolutionary conserved nucleotide positions/regions; (ii) protein function, stability, and interactions; (iii) splicing, branching, and miRNA binding sites; (iv) promoters and transcription factor binding sites; and (v) collocation with QTL. Seventy-one genetic variants were identified in the caseins (CSN1S1, CSN2, CSN1S2, and CSN3), LALBA, LGB, and LTF genes. Eleven potentially regulatory variants and two missense mutations were identified. LALBA Ile60Val was predicted to affect protein stability and flexibility, by reducing the number the disulfide bonds established. LTF Thr546Asn is predicted to generate steric clashes, which could mildly affect iron coordination. In addition, LALBA Ile60Val and LTF Thr546Asn affect exonic splicing enhancers and silencers. Consequently, both mutations have the potential of affecting immune response at individual level, not only in the mammary gland. Although laborious, this multistep procedure for classifying variants allowed the identification of potentially functional variants for milk protein genes.

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.