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

The microflora within the gastrointestinal system directly affects energy metabolism, digestive function, mucosal immune system development, and disease pathogenesis of its eukaryotic host [1–4]. This is particularly true for herbivores, including the horse, which are dependent upon fermentation by bacteria to utilize plant structural carbohydrates [4]. Therefore, a detailed knowledge of gut microflora is essential for understanding the nutritional needs of horses and the contribution of gut homeostasis to equine health. Research on bacterial communities has recently flourished with the application of next-generation sequencing (NGS) technology [5]. Studies incorporating NGS have led to the discovery of thousands of novel species (i.e., Operational Taxonomic Units [OTUs]) and elucidation of their ecological function within the gut of vertebrates [5–7]. Numerous factors including the evolutionary history of the host, age, and diet influence the diversity of gut microbes; they in turn have been implicated in a broad range of disorders including Crohn’s disease, chronic diarrhea, inflammatory bowel disease, type I diabetes, obesity, and asthma [2, 7–9].

Alterations in hindgut bacterial communities have also been associated with several equine diseases [10–17]. Excess nonstructural carbohydrates (i.e., starches, fructans, or simple sugars) that are not digested in the foregut enter the cecum and colon, where bacterial fermentation produces byproducts including lactic acid and gas, which can cause colic [4, 16, 17]. The same initiators can also lead to the development of laminitis, which often occurs subsequent to overconsumption of grain or after feeding on lush pasture rich with nonstructural carbohydrates [18–20]. Starch and oligofructose overload-induced models have revealed strong associations between onset of laminitis and proliferation of Streptococcus and Lactobacillus bacteria, with a concurrent decrease in intraluminal pH [12–15, 21–23].

Numerous studies have characterized and enumerated bacteria of the equine hindgut, primarily relying on culturing of bacteria, clone-based sequencing of Polymerase Chain Reaction (PCR) amplicons, denaturing gradient gel electrophoresis (DGGE), fluorescence in situ hybridization (FISH), or gene terminal restriction fragment length polymorphism (T-RFLP) [21, 23–32]. The primary microbes detected consisted of Gram-positive bacteria, many of which were associated with the cluster XIVa of Clostridiaceae, Streptococcus spp., and Lactobacillus spp. [14, 22, 24, 26]. Up to 96% of all observed OTUs could not be assigned, highlighting how little was known about this ecosystem [30].

Recently researchers have begun to apply 454 sequencing of 16S rRNA amplicons to understand the equine gut microflora [10, 33, 34]. A total of 1,518 OTUs have been observed in feces from just two horses, with Firmicutes, Verrucomicrobia, and Proteobacteria being the most abundant Phyla, and Subdivision 5 Incertae sedis spp., TM7 Incertae sedis spp., and Treponema spp. the most common genera [33]. In a study examining colitis, Firmicutes were found to dominate the feces of normal horses in contrast to Bacteroidetes in horses with undifferentiated colitis [10]. Bacterial communities in the stomach were also found to be dominated by the Phyla Firmicutes, Proteobacteria, and Bacteroidetes, with Lactobacillus spp., Streptococcus spp., and Moraxella spp. comprising the most abundant genera [34]. The stomach microflora segregated based on management (stabled versus pastured) and sampling methods (biopsy versus post mortem) [34]. These studies show a much more diverse assembly of bacteria than previously described; however, the mechanisms linking bacterial diversity to diseases such as colic, colitis, and laminitis are yet to be elucidated.

We thus explored the equine hindgut microflora by pyrosequencing bacterial 16S rRNA gene segments present in feces of normal horses and those suffering from chronic laminitis. Our goals were to (1) describe the level of microbial diversity and (2) compare the microflora of healthy horses to those with chronic laminitis. We hypothesized that horses with chronic laminitis, which had in the past experienced a bout of acute laminitis and presumably a radical shift in bacterial flora that accompanies this disease, would harbor a different microbial population. Our study contributes to the characterization of the equine gut microbiome and its potential link to laminitis.

We observed more unique OTUs (3,234) than detected by Shepherd et al. [33] (1,510 OTUs) despite our lower read depth (2,204 versus 28,458, respectively). This is likely because we had a greater number of horses (16 versus 2). However, our Chao1 index of bacterial richness (795.7) and Shannon Index of bacterial diversity (5.07) were lower than in the previous study (2,359 and 6.7, respectively) [33]. There was significantly higher bacterial diversity as estimated from OTUs and the Chao1 index in the laminitis group compared to the control (P = 0.019, P = 0.020, respectively). The only other significant differences between the control and laminitis groups was the higher abundance of two undescribed genera of Clostridiales in the laminitis horses (P = 0.03 and P = 0.01, respectively). This suggests potential changes in bacterial communities that should be further explored.

Our lower bacterial richness and diversity relative to what was previously reported could be attributed to an insufficient number of reads to capture all of the diversity within each sample, particularly for the low abundance OTUs [33]. This is supported by our OTU rarefaction plot that fails to plateau (Figure 1A). Future studies need to generate closer to the 5,000 reads per sample previously recommended [10]. We targeted this level of depth; however, because one of our samples was over-represented (30,911) in the pooled multiplex of amplicons, it reduced the number of reads that were generated for the other samples. Therefore, greater attention needs to be given to DNA extraction, PCR amplification, and library construction so that each amplicon is equally represented.

We successfully assigned a greater number of reads to Phyla (98.42% versus < 62%) than several previous studies using 16S rRNA sequences [24, 33]. This is likely because they did not identify and exclude chimeras, which are known to inflate the number of unclassified OTUs [35]. We detected the same number of Phyla (16) as in Shepherd et al.[33], including 4 that were not previously observed in horses; MVP-15, Synergistetes, Chlamydiae, and Deferribacteres [10, 33]. The most abundant Phylum we observed in horses, Firmicutes, was also the major component of equine intestinal flora in previous studies that analyzed feces from two adult Arabian geldings [33] and 6 healthy horses [10], stomach contents from 9 hay-fed stabled horses [34], and more traditional studies that used clone-based Sanger sequencing [24, 31]. Firmicutes are also common in the gut of other diverse taxa, from cats, dogs, and polar bears to cattle [38–40]. In contrast, Bacteroidetes was the most abundant Phylum among horses that had colitis, supporting the hypothesis that Firmicutes play an important role in gut function [10].

Verrucomicrobia, Bacteroidetes, and Proteobacteria represented the next largest components of the equine gut microbiome that we observed; a pattern similar to previous studies, although the Phyla were not always in the same Order [10, 31, 33]. We detected higher levels of Verrucomicrobia than previously reported (21.78% versus < 5%) [10, 24]. The abundance of this Phylum in horses from central Texas suggests it plays a more important role in hindgut function than previously appreciated. Our second most abundant genus among all horses was an unknown type within the RFP12 Order of Verrucomicrobia. This is a good candidate for culturing in order to classify it and characterize this taxa’s metabolic function.

The cecum and colon of the horse are important for the breakdown of structural carbohydrates and production of volatile fatty acids [4]. Therefore, we expected to detect bacteria known to play such a role, including Ruminococcus spp., Fibrobacter spp., Eubacterium spp., and Treponema spp. [24, 41]. We indeed detected all of the above; Ruminococcus had a mean of 1.03%, Fibrobacter 0.042%, Eubacterium 0.004%, and Treponema 2.18%. Our values were consistent with what has been previously observed (0.50% – 4.4%, 0.01% – 0.75%, 0.09%, 1.90% – 3.00%, respectively) [10, 24, 33, 41]. Interestingly, among the most abundant were unassigned genera of Ruminococcaceae that together composed 8.75% of all OTUs. These may represent important uncharacterized cellulytic bacteria and warrant further investigation.

A vast amount of individual variation was observed in horses at all taxonomic levels. A large portion of this likely came from environmental heterogeneity and differences in animal history, combined with lack of sequencing depth. However, similar individual variation in the equine gut microflora was previously observed. For example, in a study that had a mean of 4,712 reads per sample Bacteroidetes varied from 9.0% to 21.3% and Proteobacteria from 0.0% to 42.7% [10]. Such large individual variation may be a natural trait of equine gut communities; however, the lack of detailed studies using a large number of horse samples limits the inferences that can be made from these patterns.

The genera previously found dominating the lower intestinal microflora in two Arabian geldings based on 16S rRNA pyrosequencing of fecal samples included Blautia spp., Fibrobacter spp., Subdivision 5 Incertae sedis spp., TM7 Incertae sedis spp., Treponema spp., and Ruminococcus spp. [33]. In contrast, fecal analysis of a more diverse group of horses found the primary genera Clostridium spp., Coptotermes spp., Enterococcus spp., Fusobacterium spp., Porphyromonas spp., Pseudomonas spp., and Prevotella spp. [10]. A study that used intestinal samples detected many unassigned genera affiliated with Clostridium spp., Butyrivibrio spp., Ruminococcus spp., and Eubacterium spp. [24]. We detected all of the above except Coptotermes spp., Porphyromonas spp., and Pseudomonas spp.. Among the 14 genera that we observed with > 1.0% abundance were Streptococcus spp., Akkermansia spp., and Oscillospira spp., and 8 genera that could not be assigned to any described genus. This large proportion of unassigned genera among highly abundant OTUs highlights the need for more traditional studies characterizing bacteria and their phenotypic traits to better understand the function of the equine hindgut microflora.

Within abundant genera we found evidence suggesting additional diversity. The most diverse genus was Clostridium, which exhibits a wide range of functions and contains both beneficial and pathogenic representatives [42]. For example, C. botulinum causes botulism as well as productivity problems and C. difficile leads to severe diarrhea and colitis in both humans and livestock [43, 44]. Yet, many Clostridium spp. are cellulytic and important for the digestion of plant material [45, 46]. We detected 33 species of Clostridium, including C. botulinum in one horse. The population dynamics of bacterial species and their interactions can influence normal gut function and the development of diseases [2]. It is possible that some of the bacterial shifts that affect disease states such as laminitis occur at the species level.

There are numerous lines of evidence suggesting hindgut microflora play a role in the development of laminitis. Several studies have examined the bacterial response during various experimental laminitis models [12–14, 21–23]. An estimated 53% of acute laminitis cases occur after overconsumption of grain or grass rich with nonstructural carbohydrates (i.e., starch, fructans, or simple sugars) [20], which is also associated with an explosive proliferation of Streptococcus spp. and Lactobacillus spp. in the cecum and a concurrent decrease in the intraluminal pH [1, 12, 15]. Potentially, either of these may be a factor in laminitis. We found remarkable variation in Streptococcus spp. among healthy horses (0.40% to 91.96% of all OTUs); therefore the absolute abundance of Streptococcus spp. might not be important relative to other changes disrupting hindgut equilibrium.

In the carbohydrate overload model of laminitis, Garner et al. [23] found that Lactobacillus spp. increased in abundance by a factor of 10⁵. These changes led to decreased intraluminal pH through the production of lactic acid, which caused death and lysis of other bacterial species including Enterobacteriaceae spp. and Bacilli spp. [23]. Garner hypothesized that these release endotoxins and cause mucosal damage, contributing to the development of laminitis. Endotoxins can escape into the bloodstream and cause immune system activation, inflammation, fever, low blood pressure, and high respiration rate; some of these symptoms appear during the early stages of laminitis [47–49]. We found Lactobacillus spp. represented a small portion of the bacterial communities in the horses we sampled (0.82% controls, 0.60% laminitis). However, we only obtained samples from horses that had a previous history of this condition and not immediately after a relapse of laminitis. Therefore we would not have detected any previous transient Lactobacillus spp. proliferation. In addition, we sampled the microflora using feces, an approach which could potentially mask changes occurring in the stomach, cecum, and upper colon [32]. The effects Lactobacillus spp. and Streptococcus spp. proliferation has on the equine gut microbiome following an increase in dietary nonstructural carbohydrates and relapse of chronic laminitis should be explored.

The composition of the hindgut microflora also has large impacts on feed digestibility and equine nutrition because the horse depends upon microbial fermentation to digest plant structural carbohydrates [4]. Similar to previous studies we found that majority of the abundant bacterial genera were anaerobic fermenters, suggesting that the hindgut microflora are specialized for breaking down plant material. Alterations to bacterial communities may confer advantages to horses under certain dietary conditions [50]. For example, gradual addition of grain into the diet increases the ratio of propionate to acetate, presumably by altering the bacterial microflora [51]. Propionate can be directly converted to glucose and thus this shift is beneficial for horses with high energy needs [4]. However, grain also has more simple sugars, which increase the risk of colic and laminitis [13, 17, 21]. Future studies should explore how bacterial diversity and function can mediate adaptation to high-energy diets and reduce disease risks.

Our exploration of the equine hindgut microflora revealed higher levels of bacterial diversity in horses with chronic laminitis and identification of two Clostridiales genera that differed in abundance from control horses. We observed large individual variation suggesting that bacterial populations may be influenced by factors such as genetic background, age, diet, feeding time, and body condition, which were not taken into account during this study. There was high abundance of cellulytic bacteria, primarily Ruminococcaceae and Clostridiaceae. We observed numerous abundant uncharacterized genera within Subdivision 5 of Verrucomicrobia, Clostridiales, and Ruminococcaceae that warrant further investigation into their function. Vast individual differences in Streptococcus abundance among healthy horses suggested that this genus is likely not closely linked with chronic laminitis. We recommend studies make efforts to reduce experimental variation by using more homogenous horse populations and incorporating rigorous normalization during 454 library construction to increase the sensitivity for biologically-relevant changes in bacterial communities.