Pyrosequencing of 16S rRNA genes in fecal samples reveals high diversity of hindgut microflora in horses and potential links to chronic laminitis

BMC Veterinary Research (Impact Factor: 1.78). 11/2012; 8(1):231. DOI: 10.1186/1746-6148-8-231
Source: PubMed


The nutrition and health of horses is closely tied to their gastrointestinal microflora. Gut bacteria break down plant structural carbohydrates and produce volatile fatty acids, which are a major source of energy for horses. Bacterial communities are also essential for maintaining gut homeostasis and have been hypothesized to contribute to various diseases including laminitis. We performed pyrosequencing of 16S rRNA bacterial genes isolated from fecal material to characterize hindgut bacterial communities in healthy horses and those with chronic laminitis.

Fecal samples were collected from 10 normal horses and 8 horses with chronic laminitis. Genomic DNA was extracted and the V4-V5 segment of the 16S rRNA gene was PCR amplified and sequenced on the 454 platform generating a mean of 2,425 reads per sample after quality trimming. The bacterial communities were dominated by Firmicutes (69.21% control, 56.72% laminitis) and Verrucomicrobia (18.13% control, 27.63% laminitis), followed by Bacteroidetes, Proteobacteria, and Spirochaetes. We observed more OTUs per individual in the laminitis group than the control group (419.6 and 355.2, respectively, P = 0.019) along with a difference in the abundance of two unassigned Clostridiales genera (P = 0.03 and P = 0.01). The most abundant bacteria were Streptococcus spp., Clostridium spp., and Treponema spp.; along with unassigned genera from Subdivision 5 of Verrucomicrobia, Ruminococcaceae, and Clostridiaceae, which together constituted ~ 80% of all OTUs. There was a high level of individual variation across all taxonomic ranks.

Our exploration of the equine fecal microflora revealed higher bacterial diversity in horses with chronic laminitis and identification of two Clostridiales genera that differed in abundance from control horses. There was large individual variation in bacterial communities that was not explained in our study. The core hindgut microflora was dominated by Streptococcus spp., several cellulytic genera, and a large proportion of uncharacterized OTUs that warrant further investigation regarding their function. Our data provide a foundation for future investigations of hindgut bacterial factors that may influence the development and progression of chronic laminitis.

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    • "An effect of transportation, fasting and anaesthesia on alpha diversity indices (richness, diversity, evenness) was not apparent. Similarly, diversity indices did not differ between horses affected with colitis [3] or laminitis [5] and controls. This suggests that an overall loss of bacterial diversity might not play an equal role in equine disease development, and shifts on the species level and microbiota structure might be more important. "
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    ABSTRACT: The intestinal microbiota is important for health and disease. Factors that disturb the equine intestinal microbiota need further investigation. To determine the effects of transport, fasting and anaesthesia on the faecal microbiota of healthy adult horses using next generation sequencing. Experimental trial. Faecal samples were taken from 8 horses at baseline, after transport, 12 h of fasting and 24 h, 48 h and 72 h after a 6-h anaesthesia. Next generation sequencing of the V4 region of the 16S rRNA gene was used to assess the microbial composition of faeces. Alpha diversity, phylogenetic structures and beta diversity were assessed using MOTHUR. There were significant changes in the relative abundances of phyla, classes, orders and families after transport, fasting and anaesthesia. Most notably horses had a significantly lower abundance of Clostridiales after transport compared to baseline (p = 0.03) and a decreased abundance of Rickettsiales after fasting (p = 0.024). Alpha diversity was not significantly different between time points (all p>0.21). When parsimony analysis was applied, anaesthesia had a significant effect on community membership and structure (Jaccard and Yue & Clayton index both p = 0.02). There was some effect of transport, fasting and anaesthesia on the composition and structure of the microbiota of healthy horses. This indicates these are potentially stress factors for the equine intestinal microbiota. Further investigation is required to look at the potential impact of changes in the microbiota on the development of disease in the post-anaesthetic period. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    No preview · Article · Jun 2015 · Equine Veterinary Journal
    • "Limitations of this study should be considered. The sample size was small but consistent with other studies of the equine microbiota (Costa et al., 2012;Dougal et al., 2012;Steelman et al., 2012). The sample size could have limited statistical power and the ability to detect differences; however, numerous differences were identified . "
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    ABSTRACT: The advance of new sequencing technologies has allowed more comprehensive characterization of complex microbial communities, including the ones inhabiting the intestinal tract. The presence of extreme environmental filters, such as low pH, digestive enzymes and anaerobic conditions along the tract, acts on the selection of unique bacteria in each compartment. The intestinal microbiota has an enormous impact on the maintenance of health. However, data about the bacteria present in the different intestinal compartments of horses are sparse. In this study, high throughput sequencing was used to characterize and compare bacterial profiles from different intestinal compartments of 11 horses scheduled for euthanasia for reasons other than gastrointestinal problems. Marked differences among compartments even at high taxonomic levels were found, with Firmicutes comprising the main bacterial phylum in all compartments. Lactobacillus spp. and Sarcina spp. predominated in the stomach and a marked increase of Streptococcus spp. occurred in the duodenum. Actinobacillus and Clostridium sensu stricto were the most abundant genera in the ileum and '5 genus incertae sedis', a genus from the Subdivision 5 class of the Verrucomicrobia, was the most abundant from the large colon through feces. There was a significant increase in diversity towards the distal gut with similar profiles observed from the cecum through feces at the class level. The bacterial population comprising the equine intestinal tract varies greatly among compartments and fecal samples may be useful as representative of changes occurring in the distal compartments. Copyright © 2015 Elsevier Ltd. All rights reserved.
    No preview · Article · Mar 2015 · The Veterinary Journal
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    • "The dominance of a small number of phyla is consistent with other fecal microbiota studies, where a large number of phyla may be present with only a few accounting for more than 1% of sequences. Typically, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Verrucomicrobia are most common [14,23-26], as was the case here. The relative abundance of Bacteroidetes was low compared to studies of cattle [15,16,21]; however, care must be taken when making such comparisons and comparison of bison and cattle samples using the same laboratory and analytical methods is required to evaluate potential differences between those species. "
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    ABSTRACT: Background The intestinal tract harbours a complex and diverse microbial population that is important for health, yet has been poorly described in many species. This study explored the fecal microbiota of semi-free-ranging Wood bison (Bison bison athabascae). Results A total of 2081936 16S rRNA (V4) sequences from 40 bison were evaluated. CatchAll analysis of richness predicted a mean of 10685 species per sample (range 5428–24764, SD 4136). Diversity was high, with an average inverse Simpson’s index of 31.78 (SD 15.3, range 8.55-86.7). Twenty-one different phyla were identified; however, only Firmicutes and Proteobacteria, Actinobacteria accounted for >1% of sequences. Two distinct population clusters (Group A, n = 19 and Group B, n = 21) were evident based on both community membership and population structure. Group A had a significantly lower relative abundance of Actinobacteria (6.4 vs 11.8%, P = 0.002), Chloroflexi (0.002 vs 0.013%, P = 0.014), Gemmatimonadetes (0.007 vs 0.15%, P = 0.038) and Proteobacteria (18.7 vs 42.5%, P = <0.0001) and a greater relative abundance of Firmicutes (70.9 vs 39.3%, P < 0.0001) than Group B. Within Group B, Alphaproteobacteria was the most common class of Proteobacteria (28% of all sequences), while Caulobacteraceae (18.5%), Pseudomonadaceae (3.5%), Hyphomicrobiaceae (3.5%), Alcaligenaceae (3.1%) and Xanthomonadaceae (2.6%) were the most abundant families. The twenty (3.1%) most abundant genera accounted for 71% of sequences. No operational taxon units (OTUs) were found in all samples at a relative abundance of 1% or greater. One OTU (Clostridium cluster XI) was present at 1% or more in all Group A samples, with two other Clostridium cluster XI OTUs in 18/19 (95%) samples. No OTUs were found at that abundance in all Group B sample, but an unclassified Lachnospiraceae was present in 20/21 (95%) and Clostridium cluster XI and Brevundimonas were found in 19 (90%) samples. Conclusions The fecal microbiota of Wood bison is rich and diverse. The presence of two distinct populations not associated with housing, age or gender suggest that enterotypes, distinctly different microbial population compositions that can achieve the same ultimate function, might be present in bison, as has been suggested in humans.
    Full-text · Article · May 2014 · BMC Veterinary Research
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