Metagenomic investigation of the intestinal microbiome in healthy

Metagenomic investigation of the intestinal microbiome in healthy and
diarrheic horses
1 Dept.
L. G.
H. R.
J S.
of Pathobiology, 2 Dept. of Clinical Studies, University of Guelph, Guelph, Ontario, Canada
The intestinal tract contains one of the most dense, dynamic and complex
bacterial populations (microbiomes) of any environment on the planet. It has been
called the ‘2nd genome’ in testament to its size and complexity. In humans, it is
believed that the intestinal microbiome contains up to 1000 different species and
approximately 1012 bacteria/g.
The intestinal microbiome acts as a barrier to establishment and overgrowth
of enteropathogens, interacts with the immune system, and plays a crucial role in
digestion. It also participates in many other critical functions that are poorly
understood. In horses, disruption of the intestinal microflora is thought to be
associated with a wide range of problems, most notably colitis and laminitis. Yet,
despite the clear importance of the intestinal microflora, our understanding of
what constitutes ‘normal’ and ‘abnormal’ is woefully inadequate. Most
investigations of the equine microflora have typically involved bacterial culture,
which only allows for superficial assessment of parts of the cultivable component
on the microflora, a significant limitation since a large component on the
microflora is thought to consist of unknown or unculturable microorganisms. The
development of culture-independent methods has led to a revolution in
characterization of complex microbial populations.
The objectives of this study were to characterize the fecal microbiome in
healthy horses and compare that to horses with diarrhea of varying etiologies.
Six healthy and 10 diarrheic horses with idiopathic diarrhea were enrolled. The
number of sequences that passed all quality control filters ranged from 6579 and
21600 per horse, 12901 + 6491 (mean + SD) in healthy horses and 11834 +8550 in
diarrheic horses (P=0.63) (Table 1).
99.5% of sequences from healthy horses and 99.7% from diarrheic horses
were characterized to at least the Phylum level. The remaining sequences could
indicate novel Phyla.
In healthy horses, the Firmicutes Phylum predominated, accounting from 4973% (mean 65%, SD 12%) of sequences, while Bacteroidetes were most abundant
in diarrheic horses (Figure 1). Diarrheic horses had significantly more Fusobacteria
and Spirochaetes and fewer Actinobacteria (all P<0.0003).
54-119 different Genera were identified in healthy horses, corresponding to
100-239 different species, compared to 38-104 Genera and 70-196 Species in
diarrheic horses (P=0.14 and 0.08, respectively).
Numerous different Clostridium species were identified. In healthy horses,
16.3 + 4.4 species were present, compared to 11.5 + 5.6 in diarrheic horses
The species richness indicates the complexity of the equine intestinal
microbiome and this study provides the most comprehensive indication of this
important and complex microbiome.
Numerous differences between normal and diarrheic horses were identified,
including changes at a high (Phylum) level. Differentiating cause versus effect is
impossible, but identification of organisms disproportionately present in diarrheic
horses can lead to investigation of their potential role as causative agents.
The predominance of Clostridia and related organisms in healthy horses
demonstrates the importance of this much-maligned group of bacteria.
The decreased abundance of Firmicutes suggests that efforts at
therapeutically manipulating the intestinal microbiome may need to concentrate on
this group, rather than the typical approach using lactic acid bacteria.
The abundance of Fusobacteria in diarrheic horses was interesting as the role
of this Phylum in equine colitis has not been reported.
Various unculturable (e.g. Clostridium sordellii) and previously unidentified
organisms were identified, indicating the need for non-culture-dependent methods.
Similarly, some organisms that are uncommonly isolated from healthy horses (e.g.
Clostridium perfringens from 3/6 healthy horses) were detected.
While culture independent methods and next generation sequencing eliminate
many biases from culture or cloning-based methods, there can be some PCR
amplification bias, so certain groups (e.g. Bifidobacterium spp) might be
underestimated. Evaluation of other target genes is indicated for further
comprehensive study of this microbiome.
The marked differences in the microbiome between healthy and
diarrheic horses indicate that colitis needs to be considered a population
disease, rather than one that occurs simply through overgrowth of an
individual pathogen.
Materials and Methods
Fecal samples were collected from 6 healthy and 10 diarrheic horses
presented to the Ontario Veterinary College for treatment of idiopathic colitis.
Healthy horses had no recent (90d) history of gastrointestinal disease, antibiotic
treatment or probiotic treatment.
DNA was extracted and purified. 16s rRNA gene (V3-V5 region) PCR was
performed, followed by next generation sequencing (Roche GS Junior 454
Sequencer). Standard quality control filters were applied.
( for analysis using the SILVA Small Subunit rRNA
database, with a maximum e-value of 1e-30, minimum identify of 97%, and
minimum alignment length of 75 base pairs (bp) as cutoff values. A convenience
selection of sequences were loaded into NCBI BLAST using the nucleotide
collection (nr/nt) database to confirm sequence identity determinations.
Descriptive data were generated. Principal component analysis was
performed. Comparisons between diarrheic and normal horses were performing by
t-test or Mann-Whitney test, as appropriate. Relative abundance comparisons were
based on normalized data.
This study was supported by Equine Guelph.
Table 1: Sequence data summary.
Figure 1: Comparison of the fecal microbiome of healthy and
diarrheic horses at the Phylum level.
Figure 2: Principle component analysis of
healthy (red) and diarrheic (green).
Organisms typically associated with the oral microflora were common, such as
Capnocytophaga spp, (5/6 normal and 6/10 diarrheic, at up to 3.9% of sequences)
and Porphyromonas spp (5/6 normal and 10/10 diarrheic, up to 9.8% of
31 different Lactobacillus spp were identified, but there was no difference in
relative abundance between groups (P=0.35). Between 1-5 (mean 3.2)
Lactobacillus spp were present in healthy horses and 2-11 (mean 5.3) in diarrheic
horses (P=0.19).
Clostridium difficile was detected in 3/10 diarrheic and 1/6 healthy horses.
Escherichia coli was detected in 8/10 diarrheic but 0/6 healthy horses. Clostridium
sordellii was detected in 2 diarrheic horses.
Various organisms that are recognized enteropathogens in other species but
of unknown relevance in horses were detected, such as Yersinia and Shigella spp.
Figure 2: Relative
frequency of Phyla
diarrheic horses

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