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      Faecal microbiota characterisation of horses using 16 rdna barcoded pyrosequencing, and carriage rate of clostridium difficile at hospital admission

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          Abstract

          Background

          The equine faecal microbiota is very complex and remains largely unknown, while interspecies interactions have an important contribution to animal health. Clostridium difficile has been identified as an important cause of diarrhoea in horses. This study provides further information on the nature of the bacterial communities present in horses developing an episode of diarrhoea. The prevalence of C. difficile in hospitalised horses at the time of admission is also reported.

          Results

          Bacterial diversity of the gut microbiota in diarrhoea is lower than that in non-diarrhoeic horses in terms of species richness (p-value <0.002) and in population evenness (p-value: 0.02). Statistical differences for Actinobacillus, Porphyromonas, RC9 group , Roseburia and Ruminococcaceae were revealed. Fusobacteria was found in horses with diarrhoea but not in any of the horses with non-diarrheic faeces. In contrast, Akkermansia was among the three predominant taxa in all of the horses studied. The overall prevalence of C. difficile in the total samples of hospitalised horses at admission was 3.7 % (5/134), with five different PCR-ribotypes identified, including PCR-ribotype 014. Two colonised horses displayed a decreased bacterial species richness compared to the remaining subjects studied, which shared the same Bacteroides genus. However, none of the positive animals had diarrhoea at the moment of sampling.

          Conclusions

          The abundance of some taxa in the faecal microbiota of diarrhoeic horses can be a result of microbiome dysbiosis, and therefore a cause of intestinal disease, or some of these taxa may act as equine enteric pathogens. Clostridium difficile colonisation seems to be transient in all of the horses studied, without overgrowth to trigger infection. A large proportion of the sequences were unclassified, showing the complexity of horses’ faecal microbiota.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12866-015-0514-5) contains supplementary material, which is available to authorized users.

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          Most cited references31

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          Accurate determination of microbial diversity from 454 pyrosequencing data.

          We present an algorithm, PyroNoise, that clusters the flowgrams of 454 pyrosequencing reads using a distance measure that models sequencing noise. This infers the true sequences in a collection of amplicons. We pyrosequenced a known mixture of microbial 16S rDNA sequences extracted from a lake and found that without noise reduction the number of operational taxonomic units is overestimated but using PyroNoise it can be accurately calculated.
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            Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut.

            The human intestinal tract harbors a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the bacterial diversity (both cultivated and noncultivated bacteria) within an adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were classified into 82 molecular species (at least 98% similarity). Three phylogenetic groups contained 95% of the clones: the Bacteroides group, the Clostridium coccoides group, and the Clostridium leptum subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Only 24% of the molecular species recovered corresponded to described organisms (those whose sequences were available in public databases), and all of these were established members of the dominant human fecal flora (e.g., Bacteroides thetaiotaomicron, Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms and clearly derived from hitherto unknown species within this human gut microflora.
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              Comparison of the Fecal Microbiota of Healthy Horses and Horses with Colitis by High Throughput Sequencing of the V3-V5 Region of the 16S rRNA Gene

              The intestinal tract houses one of the richest and most complex microbial populations on the planet, and plays a critical role in health and a wide range of diseases. Limited studies using new sequencing technologies in horses are available. The objective of this study was to characterize the fecal microbiome of healthy horses and to compare the fecal microbiome of healthy horses to that of horses with undifferentiated colitis. A total of 195,748 sequences obtained from 6 healthy horses and 10 horses affected by undifferentiated colitis were analyzed. Firmicutes predominated (68%) among healthy horses followed by Bacteroidetes (14%) and Proteobacteria (10%). In contrast, Bacteroidetes (40%) was the most abundant phylum among horses with colitis, followed by Firmicutes (30%) and Proteobacteria (18%). Healthy horses had a significantly higher relative abundance of Actinobacteria and Spirochaetes while horses with colitis had significantly more Fusobacteria. Members of the Clostridia class were more abundant in healthy horses. Members of the Lachnospiraceae family were the most frequently shared among healthy individuals. The species richness reported here indicates the complexity of the equine intestinal microbiome. The predominance of Clostridia demonstrates the importance of this group of bacteria in healthy horses. The marked differences in the microbiome between healthy horses and horses with colitis indicate that colitis may be a disease of gut dysbiosis, rather than one that occurs simply through overgrowth of an individual pathogen.
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                Author and article information

                Contributors
                c.rodriguez@ulg.ac.be
                bernard.taminiau@ulg.ac.be
                bastien.brevers@student.hepl.be
                veronique.avesani@uclouvain.be
                johan.vanbroeck@uclouvain.be
                aurelia.leroux@ulg.ac.be
                marjonneur@hotmail.com
                bruwierantoine@hotmail.com
                helene.amory@ulg.ac.be
                michel.delmee@uclouvain.be
                georges.daube@ulg.ac.be
                Journal
                BMC Microbiol
                BMC Microbiol
                BMC Microbiology
                BioMed Central (London )
                1471-2180
                16 September 2015
                16 September 2015
                2015
                : 15
                : 181
                Affiliations
                [ ]Food Science Department, FARAH, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium
                [ ]Microbiology Unit, Catholic University of Louvain, Brussels, Belgium
                [ ]Equine Teaching Hospital, Clinical Department of Companion Animals and Equids, FARAH, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium
                Article
                514
                10.1186/s12866-015-0514-5
                4573688
                26377067
                9542463d-a752-4756-9f16-e543f0363d9a
                © Rodriguez et al. 2015

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 25 March 2015
                : 8 September 2015
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2015

                Microbiology & Virology
                Microbiology & Virology

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