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      Vaginal Microbiome Characterization of Nellore Cattle Using Metagenomic Analysis

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          Abstract

          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.

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          Most cited references34

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          Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses.

          Although the applicability of small subunit ribosomal RNA (16S rRNA) sequences for bacterial classification is now well accepted, the general use of these molecules has been hindered by the technical difficulty of obtaining their sequences. A protocol is described for rapidly generating large blocks of 16S rRNA sequence data without isolation of the 16S rRNA or cloning of its gene. The 16S rRNA in bulk cellular RNA preparations is selectively targeted for dideoxynucleotide-terminated sequencing by using reverse transcriptase and synthetic oligodeoxynucleotide primers complementary to universally conserved 16S rRNA sequences. Three particularly useful priming sites, which provide access to the three major 16S rRNA structural domains, routinely yield 800-1000 nucleotides of 16S rRNA sequence. The method is evaluated with respect to accuracy, sensitivity to modified nucleotides in the template RNA, and phylogenetic usefulness, by examination of several 16S rRNAs whose gene sequences are known. The relative simplicity of this approach should facilitate a rapid expansion of the 16S rRNA sequence collection available for phylogenetic analyses.
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            The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species.

            Lactobacillus acidophilus has been reported to be the predominant vaginal species. Vaginal lactobacilli isolated from 215 sexually active women were identified using whole-chromosomal DNA probes to 20 American Type Culture Collection Lactobacillus strains. Most women were colonized by L. crispatus (32%), followed by L. jensenii (23%), a previously undescribed species designated L. 1086V (15%), L. gasseri (5%), L. fermentum (0.3%), L. oris (0.3%), L. reuteri (0.3%), L. ruminis (0.3%), and L. vaginalis (0.3%). H2O2 was produced by 95% of L. crispatus and 94% of L. jensenii isolates, compared with only 9% of L. 1086V. Colonization by L. crispatus or L. jensenii was positively associated with being white (P /=20 years (P=.05), barrier contraceptive usage (P=.008), and lower frequency of bacterial vaginosis (P<.001) and gonorrhea (P=.03). L. crispatus and L. jensenii, not L. acidophilus, are the most common species of vaginal lactobacilli.
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              Taxonomic identification of commensal bacteria associated with the mucosa and digesta throughout the gastrointestinal tracts of preweaned calves.

              Bacterial colonization in the gastrointestinal tracts (GIT) of preweaned calves is very important, since it can influence early development and postweaning performance and health. This study investigated the composition of the bacteria along the GIT (rumen, jejunum, ileum, cecum, and colon) of preweaned bull calves (3 weeks old) using pyrosequencing to understand the segregation of bacteria between the mucosal surface and digesta. Phylogenetic analysis revealed that a total of 83 genera belonging to 13 phyla were distributed throughout the GIT of preweaned calves, with the Firmicutes, Bacteroidetes, and Proteobacteria predominating. Quantitative PCR (qPCR) analysis of selected abundant bacterial genera (Prevotella, Bacteroides, Lactobacillus, and Faecalibacterium) revealed that their prevalence was significantly different among the GIT regions and between mucosa- and digesta-associated communities. Rumens contained the most diverse bacterial population, consisting of 47 genera, including 16 rumen-specific genera, followed by the large intestine and then the small intestine. Bacterial species richness was higher at the mucosal surface than in the local digesta, with the exception of the rumen. The majority of bacteria found on the rumen epithelial surface and within the small intestine could not be identified due to a lack of known genus-level information. Thus, future studies will be required to fully characterize the microbiome during the development of the rumens and the mucosal immune systems of newborn calves. This is the first study to analyze in depth the bacterial composition of the GIT microbiome in preweaned calves, which extends previous findings regarding early rumen colonization and bacterial segregation between mucosa- and digesta-associated microbial communities.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                24 November 2015
                2015
                : 10
                : 11
                : e0143294
                Affiliations
                [1 ]Departmento de Microbiologia, Instituto de Ciencias Biologicas, Universidade Federal Minas Gerais, Belo Horizonte, MG, Brazil
                [2 ]René Rachou Research Center – Fiocruz, Belo Horizonte, MG, Brazil
                Cairo University, EGYPT
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MLN JRN GCO EFBS. Performed the experiments: MLN KMGRB MRG SGF FMGA ACMS. Analyzed the data: MLN LRL. Contributed reagents/materials/analysis tools: JRN GCO EFBS. Wrote the paper: MLN JRN GCO EFBS.

                Article
                PONE-D-15-35174
                10.1371/journal.pone.0143294
                4657983
                26599789
                5a562761-5fb5-4896-b59f-de0084cae682
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 10 August 2015
                : 3 November 2015
                Page count
                Figures: 9, Tables: 1, Pages: 19
                Funding
                This work was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq grant 473879/2013-1) EFBS ( http://www.cnpq.br/); Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq grant 309312/2012-4) GCO ( http://www.cnpq.br/); Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG Grant RED-00014-14) GCO ( http://www.fapemig.br/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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