on August 11, 2019 by guest · 36 mainly Streptococcus spp., Lactobacillus spp. and Sarcina spp . Cluster 2 was more diverse, with 37 predominantly Proteobacteria, Bacteroidetes and

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The equine stomach harbors an abundant and diverse mucosal microbiota 1

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Running title: Bacterial diversity in equine gastric mucosa 3

Keywords (5): equine; gastric; ulcer; bacteria; 16S rDNA, FISH 4

Journal Section: Microbial Ecology 5

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G.A. Perkins1§, H. C. den Bakker2§*, A.J. Burton1, H.N. Erb3, S.P. McDonough4, P.L. McDonough3, 7

J. Parker1, R. L. Rosenthal1, M. Wiedmann2, S. E. Dowd5, and K.W. Simpson1 8

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§These authors equally contributed to this study 10

1Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY. 11

2Department of Food Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, 12

NY. 13

3Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, 14

Cornell University, Ithaca, NY. 15

4Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, 16

NY. 17

5Research and Testing Laboratory, Lubbock, Texas, USA 18

*Address correspondence to: Henk C. den Bakker, Department of Food Science, College of 19

Agriculture and Life Sciences, Cornell University, Ithaca, NY. 14853. phone (607) 255-1263, fax 20

(607) 254-4868 , e-mail [email protected] . 21

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.06252-11 AEM Accepts, published online ahead of print on 3 February 2012

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Abstract 23

Little is known about the gastric mucosal microbiota in healthy horses, and its role in gastric 24

disease has not been critically examined. The present study used a combination of 16SrRNA 25

bacterial tag-encoded pyrosequencing (bTEFAP) and fluorescence in situ hybridization (FISH) to 26

characterize the composition and spatial distribution of selected gastric mucosal microbiota of 27

healthy horses. Biopsies of the squamous, glandular, antral, and any ulcerated mucosa were 28

obtained from 6 healthy horses by gastroscopy and from 3 horses immediately post-mortem. 29

Pyrosequencing was performed on biopsies from 6 of the horses and yielded 53,920 reads in total 30

with 631-4345 reads in each region per horse. The microbiome segregated into two distinct 31

clusters comprised of horses that were stabled, fed hay, and sampled at post-mortem (Cluster 1) 32

and horses that were pastured on grass, fed hay, and biopsied gastroscopically after a 12-h fast 33

(Cluster 2). The types of bacteria obtained from different anatomic regions clustered by horse 34

rather than region. The dominant bacteria in Cluster 1 were Firmicutes (>83% reads/sample)--35

mainly Streptococcus spp., Lactobacillus spp. and Sarcina spp. Cluster 2 was more diverse, with 36

predominantly Proteobacteria, Bacteroidetes and Firmicutes, consisting of Actinobacillus spp. 37

Moraxella spp., Prevotella spp. and Porphyromonas spp. Helicobacter spp. sequences were not 38

identified in any of 53,920 reads. FISH (n=9) revealed bacteria throughout the stomach in close 39

apposition to the mucosa, with significantly more Streptococcus spp. present in the glandular 40

region of the stomach. The equine stomach harbors an abundant and diverse mucosal microbiota 41

that varies by individual. 42

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Introduction 44

Historically, the stomach has been considered (based on microbial culture alone) a harsh 45

environment colonized by a relatively small number of acid-tolerant bacteria such as 46

Helicobacter spp. The clear association between Helicobacter pylori infection and development 47

of gastric inflammation, ulceration, and cancer in humans has reinforced this notion(17, 37). 48

However, the development of culture-independent molecular methods has dramatically changed 49

our perception of the enteric microbiota (20, 25). A diverse and unique community of bacteria 50

belonging to the phyla of the Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes exists 51

in the human stomach(11, 35). Fluorescence in situ hybridization (FISH; with probes against 52

bacterial 16S rDNA), has enabled characterization of the spatial distribution of gastric 53

colonization in a variety of species(49, 57). 54

The most common equine gastric disease is gastric ulceration while gastric impactions 55

and neoplasia are reported infrequently(10, 12, 30, 56, 59). Gastric ulceration affects between 53 56

to 90% of adult horses and has been associated with colic, weight loss, and decreased 57

performance(38, 61). Many (81%) outwardly healthy Thoroughbreds in race training also have 58

ulcers(61). The specific cause of equine gastric ulcers is not fully elucidated, although several 59

factors play a role. Increased gastric acidity coupled with decreased mucosal defense via a 60

compromised “gastric mucosal barrier” are major contributors. Reduced intra-gastric pH is 61

attributed to increased secretion of hydrochloric acid plus bacterial fermentation of grain to 62

volatile fatty acids(5-8, 43, 46, 47). Nearly 80% of ulcers in adult horses occur in the squamous 63

region—usually, close to the margo plicatus(9). Non-steroidal anti-inflammatory drugs 64

(NSAIDs) and corticosteroids contribute to ulceration of the glandular portion by altering the 65

gastric mucosal barrier via prostaglandin inhibition. The antral mucosa at the entrance to the 66


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duodenum is susceptible, but ulceration of the fundic glandular region is much less common(44). 67

Relatively little is known about the gastric mucosal microbiota in healthy horses, and its 68

role in gastric disease has not been critically examined using contemporary methodologies. The 69

equine stomach is not sterile; bacteria have been visualized on the surface of the non-glandular 70

and glandular equine stomach mucosa(3, 15, 28, 60, 62). Products of bacterial fermentation 71

(such as lactic acid and volatile fatty acids) have been isolated from the equine gastric contents, 72

and many anaerobes have been cultured. The anaerobes are comprised mostly of Lactobacilli, 73

Streptococci and lactatic acid bacteria of which specifically Lactobacillus salivarius, L. 74

crispatus, L. reuteri, L. mucosae, L. delbrueckii and L. agilis were identified using culture-75

independent technologies(2, 28, 62). Helicobacter-like organisms were seen in the gastric 76

mucosa of 6/15 horses sampled(16) and Helicobacter-like DNA has been detected in the gastric 77

mucosa of horses with and without ulcers(14). However, using FISH, gastric Helicobacter spp. 78

were not identified in 36 horses with antral pathology(28). These contradictory findings indicate 79

that further study is required to resolve the potential role of Helicobacter spp. in equine gastric 80

ulcers. 81

The present study used a combination of 16S rRNA bacterial tag-encoded 82

pyrosequencing (bTEFAP) and FISH to characterize the composition and spatial (anatomic) 83

distribution of the gastric mucosal microbiota of healthy horses. 84

85

MATERIALS AND METHODS 86

Horses and gastric mucosal sample collection. Gastric mucosal biopsies were obtained from 87

nine horses and their management and signalment are shown in Table 1. None of the horses had 88

clinical evidence of gastrointestinal disease or had received any non-steroidal anti-inflammatory 89


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drugs, corticosteroids, or gastroprotectants within one month prior to gastric biopsy. The 90

experiment was approved by the Cornell University Institutional Animal Care and Use 91

Committee. 92

Post-mortem gastric biopsies were obtained from three (of the nine total) non-fasted 93

horses within 30 min of euthanasia using sterile 6-mm punch biopsy instruments. Biopsies from 94

each horse and each stomach region were done using a new biopsy instrument to avoid potential 95

carryover of microbial DNA. These three horses had been used for student lameness 96

examinations including perineural and joint anesthesia plus diagnostic imaging. For 2 wks prior 97

to euthanasia, these horses were stabled and received only free-choice hay (Table 1). 98

Gastroscopic biopsies were obtained from the other six horses, which were kept at pasture 99

and supplemented with hay (Table 1). The biopsies were obtained after a 12-h fast and sedation 100

with detomidine (Dormosedan, Pfizer Animal Health, Exton, PA, USA) using a 3-m gastroscope 101

(Olympus CV-100 video endoscope) and 3-mm sterile biopsy forceps (Olympus of America, 102

Melville, New York, USA). Separate sterilized biopsy forceps were used on each horse. Forceps 103

were decontaminated between stomach regions by serial washes in 6.15% sodium hypochlorite 104

bleach and sterile 0.9% saline to reduce the possibility of microbial DNA carryover between the 105

individual samples taken from a horse. The endoscope was sterilized between horses with 55% 106

glutaraldehyde. 107

Macroscopic evaluation of the gastric mucosa was performed at post-mortem examination 108

or during endoscopy with a number and severity score(36) assigned to any gastric ulcers present 109

(Table 1). Gastric biopsies were obtained from the squamous, glandular, and antral regions and 110

from any ulcerated mucosae of each horse. All horses had two biopsies per region were placed in 111

formalin for FISH, fixed overnight, and paraffin-embedded for standard histopathology; two other 112


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biopsies per region were placed in tissue lysis (ATL) buffer (Qiagen Inc., Valencia, California, 113

USA) for DNA extraction. 114

Histopathology. Formalin-fixed paraffin-embedded sections of gastric mucosa stained with 115

haemotoxylin and eosin (H&E) and Masson’s Trichrome were ‘blindly’ evaluated for the 116

presence or absence of ulceration and gastritis by one pathologist (co-author S.P.M.). The 117

presence of bacteria was qualitatively evaluated in Gram and modified-Steiner stained sections. 118

Pyrosequencing. DNA was extracted from mucosal biopsies of all regions of the stomach from 119

six horses (H2, H3, H35, H60, H79 and H86; Table 1) using the QIAamp tissue kit (Qiagen Inc., 120

Valencia, California, USA) according to the manufacturer’s instructions. Three horses (H1, 121

H131, and H158) did not have bTEFAP performed due to inadequate or lost samples. The data 122

processing were performed as described previously, with minor alterations(26, 31, 52). The 123

bTEFAP sequencing was performed on the GS FLX Titanium sequencing platform (Roche 124

Applied Science, Indianapolis, IN). Primers 28F 5’GAGTTTGATCNTGGCTCAG and 519r 5’ 125

GTNTTACNGCGGCKGCTG were used to sequence an approximate 500 bp fragment of the 126

16S rDNA (Research and Testing Laboratory, Lubbock, TX). Raw data from bTEFAP was 127

screened and trimmed based upon Q20 phred based quality scoring and binned into individual 128

biopsy sample collections. Sequence collections were depleted of chimeras using B2C2 129

(http://www.researchandtesting.com/B2C2.html). The resulting files were then depleted of short 130

reads (< 200bp), tags, primers, non-bacterial ribosome sequences (mitochondria, plastid etc), 131

sequences with degenerate bases, sequences with >6 homopolymer stretches. The bacterial taxa 132

were then identified using BLASTn comparison to a curated high-quality 16S rDNA database 133

derived from NCBI (http://www.ncbi.nlm.nih.gov/). Relative percentages of bacteria at each 134

taxonomic level were determined for each individual biopsy sample. Data were also compiled at 135


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each individual taxonomic level according to the NCBI taxonomy criteria as described 136

previously(18, 19). Collection and sequence information is deposited at MG-RAST 137

(http://metagenomics.anl.gov/) under accession numbers 4468640.3- 4468664.3. 138

Bacterial diversity indices were performed on trimmed, non-ribosomal sequence-139

depleted, chimera-depleted, high-quality reads as described previously(1). Multiple-sequence 140

alignment was done using MUSCLE (21) (with parameter -maxiters 1, -diags1 and -sv). Based 141

on the alignment, a distance matrix was constructed using DNAdist from PHYLIP version 3.6 142

with default parameters(23). These pairwise distances served as input to DOTUR(51) for 143

clustering the sequences into operational taxonomic units (OTUs) of defined sequence similarity 144

ranging from 0% to 20% dissimilarity. From the literature, we can expect that 0% and even 3% 145

dissimilarity in sequences generated from pyrosequencing (based upon rarefaction) will provide 146

dramatic overestimation of the phylotypes present in a sample(32, 50). At 5 % dissimilarity 147

(roughly genus level classification), we expect to obtain a more accurate estimation of 148

comparative diversity present across the samples. Although such estimates have limitations, the 149

comparison among samples that were treated using similar procedures was felt to be valid yet 150

noting that some overestimation or underestimation is likely. The clusters based upon 151

dissimilarity of 3% and 5% served as OTUs for generating predictive rarefaction models and for 152

making calculations with the richness (diversity) estimators and Chao1(13). Correlation between 153

the numbers of OTUs and estimated number of OTUs was calculated in the R-package 154

(http://www.R-project.org). 155

A hierarchical cluster analysis was performed using the PVClust software (55) available 156

in the R-package (http://www.R-project.org). The data used for the cluster analysis were the 157

relative-abundance data on the genus level, using both the correlation distance option and the 158


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average cluster method. Approximately unbiased P values as computed by the PVClust 159

software(55) were reported to determine the significance of the clusters found. Principal 160

Component Analyses (PCA) and Principal Coordinate Analysis (PCoA) were performed in the 161

ape package version 2.8 (48) in the R-package. The PCoA was based on a Bray-Curtis 162

dissimilarity matrix produced in the vegan package (version 1.17-12, Oksanen et al., 163

[http://CRAN.R-project.org/package=vegan]). To determine the significance of the effect of 164

horse and stomach region on the microbiota, a permutation test for constrained correspondence 165

analysis was performed in vegan package. 166

To determine which phylotypes were most abundant within a sample, representatives of 167

the top five most abundant reads were compared to type strains in the RDP database (version 168

10.19; http://rdp.cme.msu.edu/). The choice to limit the analyses to the top five most abundant 169

reads was arbitrarily and chosen to limit the amount of data analyzed. To get a better 170

understanding of the phylogenetic placement of these sequences we also performed phylogenetic 171

comparisons of these representatives of the top five most abundant reads to type strains in the 172

RDP database. Reads were aligned to the type strains of selected groups using the existing RDP 173

alignment. Phylogenetic inferences were performed in RaxML version 7.0.4 (53) with 1000 174

rapid bootstrap replicates to infer robustness of the individual clades. The most abundant reads 175

were extracted from the data sets using tools available in the RDP pyrosequencing pipeline 176

(http://pyro.cme.msu.edu/). 177

Fluorescence in situ hybridization (FISH). The number and spatial distribution of mucosal 178

bacteria in all nine horses and gastric regions (Table 1) was evaluated by FISH with labeled 179

oligonucleotide probes directed against bacterial 16S rRNA as previously described(29). 180

Sections were hybridized with a probe that recognizes the vast majority of bacteria (EUB-338: 181


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GCTGCCTCCCGTAGGAGT labeled with 6-FAM) in combination with a probe directed 182

against Streptococcus spp. (STREP: CACTCTCCCCTTCTGCAC labeled with Cy3)(58). There 183

also was a probe directed against Lactobacillus spp. (LAB-158: 184

GGTATTAGCA(C/T)CTGTTTCCA labeled with Cy3)(24). The specificity of hybridization 185

was controlled by the combined application of an irrelevant probe (non-EUB338: 186

ACTCCTACGGGAGGCAGC) and the inclusion of control slides spotted with a broad spectrum 187

of bacteria(29). Hydridized samples were washed in phosphate buffered saline (PBS), air-dried, 188

and mounted with ProLong® Antifade Kit. Sections were examined with an Olympus BX51 189

epifluorescence microscope, and images captured with an Olympus DP-70 camera and analysis 190

system (Olympus America, PA, USA). The number of bacteria located in the free mucus, 191

adherent mucus, superficial epithelium, glandular epithelium, glandular mucus or that were 192

invasive, were determined using counts from ten representative 60x fields of view and expressed 193

as bacteria/mm2. The same person (co-author A.J.B.) performed all bacterial localization and 194

counting. 195

Non-parametric comparisons were used on the quantitative counts of bacteria/mm2 (derived 196

from FISH) for each stomach region as follows. Friedman’s test was used to compare the 197

localization (“depth”) of bacteria within the mucosa of the various stomach regions and for 198

comparison of the number of each type of bacteria between the different regions (squamous, antral, 199

and glandular), all controlled for horse. Significance was set at P ≤ 0.05 but with a Bonferroni 200

correction (for three stomach regions or for three categories of bacteria), considering any test-wise 201

(2-sided) P value ≤ 0.0167 significant. All analyses were performed using Statistix 8 v.8.0 202

(Analytical Software, Tallahassee, Florida, USA). 203


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204

Results 205

Gastric morphology. The stomach was macroscopically abnormal in five out of nine horses 206

with one horse (H3) having a grade-2 ulcer present in the squamous portion adjacent to the 207

margo plicatus, along the greater curvature at necropsy (Table 1, Fig. 1a). On histopathology, 208

this grade-2 ulcer had marked hyperplasia and parakeratosis with moderate neutrophilic infiltrate 209

and abundant adherent bacteria. The other four horses with macroscopic lesions had multiple 210

(grade 2-3; number score) small pin-point (grade-1; severity score) ulcers about 2-3 mm in 211

diameter in the squamous mucosa adjacent to the margo plicatus at the cardiac region in four 212

horses (Fig. 1b). Histopathologically, the pin-point ulcers seen endoscopically were confirmed 213

to be ulcers in only two horses (H35 and H158; Fig. 1c and d). One horse (H79) was normal 214

with parakeratosis of the mucosa at this site, while the other (H86) had a mild erosion (Table 1). 215

Endoscopic biopsies taken from the squamous region in five horses were too small (consisting 216

only of superficial parakeratosis or epidermis) for objective evaluation. The gastric mucosa 217

revealed bacteria on all sections that were predominantly Gram-positive cocci and bacterial 218

colonization appeared particularly dense in ulcerated mucosa (Fig. 1c and d). 219

Cluster analysis of the equine gastric mucosal microbiome based on relative abundance of 220

bacterial genera shows two distinct clusters . Pyrosequencing yielded 72,283 total sequences, 221

after quality control, 53,920 sequences were utilized for analysis with a range from 641 to 5345 222

reads in each sample in the six horses analyzed (Table 2). No significant correlation (Pearson’s 223

correlation, P > 0.35) was found between the number of reads per sample and the number of 224

OTUs and the estimated number of OTUs, and hence we conclude that read coverage per sample 225

does not influence estimation of phylotype diversity and richness. 226


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Analyses of the relative abundance of individual bacterial genera showed two distinct 227

clusters with a significant approximately unbiased P-value (P>0.95) (Fig. 2); (i) cluster 1 228

consisted of samples from horses H2, H3 and H86, and cluster 2 of samples from horses H35, 229

H60, H79 and H86. These clusters segregated by groups which differed in sampling method 230

(fasted and non-fasted; endoscopically and postmortem) and management practice with 231

differences including but not limited to feeding practices, pasture turn-out, exercise and 232

socializing. Specific known differences are listed in Table 1. The microbiomes did not cluster by 233

gastric region, however twelve out of twenty-two samples clustered by horse --indicating more 234

similarity in the relative abundance and taxonomic composition of the microbiota of different 235

stomach regions within an individual horse than between different horses. While there was 236

generally no significant (P < 0.95) approximately unbiased P value supporting this clustering by 237

horse, a constrained correspondence analysis (CCA) further confirmed that the horse from which 238

a sample is derived has a significant effect (ANOVA-like permutation test, P< 0.001) on the 239

microbial community, while the effect of stomach region is not significant (ANOVA-like 240

permutation test, P=0.561). Samples taken from horse H86 did not cluster exclusively in one of 241

the two clusters; samples from the glandular, antral and ulcerated regions segregated throughout 242

cluster 2, whereas the squamous region of H86 segregated into cluster 1. Differences in bacterial 243

diversity between cluster 1 (horses H2 and H3) and cluster 2 (horses H35, H60, H79 and H86) 244

were most obvious at the genus (5% divergence) level: The average number of OTUs at the 5% 245

divergence level for cluster 1 was 78 OTUs versus 128 OTUs in cluster 2. A similar pattern was 246

seen at the species level (3% divergence level). The ACE and ChaoI diversity estimators showed 247

similar patterns of a higher microbial diversity in cluster 2 than in cluster 1. The estimated 248

diversity was generally estimated to be between 3 and 25% higher than the observed number of 249


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OTUs. This suggests that the real diversity of OTUs of the individual samples is potentially 250

higher than the observed diversity. 251

Principal component analysis of the relative abundance of individual bacterial genera 252

level showed that the variation in the data (>80%) could be explained by the first two principal 253

components (Fig. 3). This analysis also confirmed the subdivision of the samples into two main 254

clusters, a cluster consisting of samples obtained from horse H2 and H3 (cluster 1) and a cluster 255

containing samples obtained from horses H35, H60, H79 and all but one sample from horse H86 256

(cluster 2). The microbiota of the sample obtained from the squamous region of horse 86 is more 257

similar to samples of cluster 1. A limited number of genera were responsible for the variance 258

explained by component 1 and 2; high abundance of Actinobacillus, Moraxella, Prevotella, 259

Porphyromonas, and to a lesser extent Gemella and Veillonella was associated with cluster 2, 260

while Lactobacillus, Streptococcus and Sarcina were associated with cluster 1. The remainder 261

of the principal component analysis only explained a variance in the data of 7% or less, and none 262

of these components could be associated to factors that were not correlated to the subdivision of 263

cluster 1 and 2, such as ulcer grade. Principal coordinate analyses of the relative abundance data 264

on the genus level revealed the same subdivision of the samples into two clusters (Fig 4). In 265

addition, this analysis also showed that samples taken from different regions in an individual 266

horse are more similar to each other than they are to samples of other horses, with the exception 267

of horse 86. 268

Comparison of the microbiota on the phylum level (Fig. 5) further shows that cluster 1 269

and cluster 2 (as found in the cluster analysis based on data on the genus level) differed in the 270

composition and diversity of their microbiota. The dominant phyla in cluster 1 were Firmicutes 271

(>83% reads/sample) whereas cluster 2 was more diverse with three main phyla: Proteobacteria 272


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(31 to 68% of the reads per sample), Bacteroidetes (6 to 44% of reads per sample) and 273

Firmicutes (15 to 42% of reads per sample). 274

A phylogenetic analysis of the top five most abundant reads showed further differences in 275

the microbial composition of samples within a cluster and within individual samples (see Table 3 276

for an overview of the single most abundant read types per sample). The most abundant read 277

types in horses 35, 60, 79 and 86 could be phylogenetically assigned to Eubacterium, 278

Actinobacillus, Moraxella, Acinetobacter and Veillonella species. Two of three samples of horse 279

H2, were found in a sistergroup to the 16S sequences of the type strains of Lactobacillus jensenii 280

and L. fornicalis (Bootstrap value [BS] 77). In contrast, the most abundant read type in the 281

squamous region of horse H2 was found in a clade with the type strains of Sarcina maxima and 282

S.ventriculi (BS 90). The most abundant read type found in two samples (antrum and glandular) 283

from horse H3 are phylogeneticaly close to the type strain of the horse-associated species 284

Lactobacillus hayakitensis, and are found in a moderately supported cluster (BS 77) together 285

with the type strain in the phylogenetic analysis. The most abundant read type in the squamous 286

region of horse H3 was a Streptococcus species, however these reads did not cluster with a 287

specific type strain. The most dominant read type in the ulcerated region of horse H3 was found 288

in a well-supported (BS 87) cluster with another horse-associated species, Lactobacillus 289

equigenerosi(22, 42). Although Helicobacter spp. have been reported to be present in the horse 290

stomach,(14, 16) none of the 53,920 reads matched this genus--indicating the absence of 291

Helicobacter spp. in our samples. FISH demonstrates gastric colonization by mucosal bacteria. 292

293

FISH analysis was performed with probes directed against all bacteria (eubacterial, EUB-338), 294

Lactobacillus spp. (LAB-158) and Streptococcus spp. (STREP). An abundant mucosal microbiota 295


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was present in all regions of the gastric mucosa examined (Fig. 6). For all nine horses, the glandular 296

region had significantly more Streptococcus sp. (P = 0.0098) compared to the squamous and antral 297

regions (Fig. 7). However, neither Lactobacillus sp. (P = 0.31) nor total bacteria (P = 0.034) 298

differed by region. Horse H3 (with the grade-2 ulcer) had the most bacteria at the ulcer site (30,530 299

bacteria/mm2; Fig. 7). When looking at specific localization (“depth”) of bacteria within the 300

mucosa, in all regions and for the Streptococcus and eubacterial probes, significantly more 301

bacteria were located in the tightly adherent mucus, followed by the free mucus and the 302

superficial epithelium (all p ≤ 0.015, Friedman’s test). The same was true for the glandular 303

region and ulcers for the Lactobacillus probe (both P ≤ 0.014) but the P values for the squamous 304

(P = 0.055) and antral regions (P = 0.025) indicated no significant variation in Lactobacillus spp. 305

numbers by “depth”. Deeply invasive bacteria were restricted to the ulcerated mucosa. 306

307 Discussion 308

309

Little is known about the gastric mucosal microbiota in healthy horses and its role in gastric 310

disease has not previously been examined. Only 30% of the fecal microbiota in people is considered 311

cultivable, and advances in molecular microbiology have revealed important variation in the 312

microbiota between different gastrointestinal segments, and in luminal contents versus mucosa in 313

healthy individuals(20, 25, 27, 33). In the present study we used a combination of culture-314

independent approaches, 16S rRNA bacterial tag encoded pyrosequencing (bTEFAP) and FISH to 315

characterize the composition and spatial distribution of the gastric mucosal microbiota of healthy 316

horses. The results of our analyses show that (i) Firmicutes, Proteobacteria, Bacteroidetes are the 317

dominant bacterial phyla in the equine gastric mucosa in this set of horses, (ii) based on the 318

taxonomic composition of the gastric microbiota, this set of horses can be subdivided into two 319


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groups, (iii) certain species of Lactobacillus, that have been previously associated with equine fecal 320

matter, are also present in the gastric mucosa of the horses, (iv) in particular Lactobacillaceae and 321

Steptococcaceae form a distinct association with the gastric mucosa, and (v) Helicobacter is not a 322

abundant taxon in the microbiota of healthy horses. 323

324

Firmicutes, Proteobacteria, Bacteroidetes are the dominant bacterial phyla in the equine 325

gastric mucosa. This culture-independent approach enabled us to identify Firmicutes, 326

Proteobacteria, Bacteroidetes as the dominant bacterial phyla in the equine gastric mucosa in this set 327

of horses. These phyla were also identified as the dominant phyla in the human gastric 328

environment(11), and mammalian gut microbiota(34), suggesting that these phyla represent the 329

dominant bacterial phyla in the mammalian gastro-intestinal tract. While the human stomach 330

environment seems to be dominated by Proteobacteria(11), the dominant phylum seems to differ by 331

sample and horse, Firmicutes being the most dominant phylum in all samples in horse H2 and H3, 332

and samples from the squamus and antral regions of horse H86, while Proteobacteria were dominant 333

in samples of horse H35 and H60 and the glandular and erosion samples of horse H86 (Fig 3). 334

Bacteroidetes, while not necessarily being the dominant phylum, made up a large proportion of the 335

microbiota found in horse H79. The composition of the gastric mucosal microbiota therefore seems 336

to differ substantially per horse. 337

338

Based on the taxonomic composition of the gastric microbiota, gastric samples of horses can 339

be subdivided into two groups. Cluster analyses and factorial analyses (both principal 340

components analysis and principal coordinate analysis) of the composition of the samples at the 341

genus level show that the samples cluster into two groups; (i) a group (cluster 1) consisting of 342


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samples dominated by Firmicutes, in particular the genera Lactobacillus , Streptococcus and 343

Sarcina, and (ii) a group (cluster 2) consisting of samples dominated either by Proteobacteria (in 344

particular Actinobacillus and Moraxella) or Bacteroidetes (Prevotella and Porphyromonas). We 345

found that this subdivision overlapped remarkably well with the way the samples were obtained 346

from the horses and the husbandry of the horses (the only exception being horse 86). For example, 347

horses in cluster 1 that were stabled, fed hay, and sampled at post-mortem, while horses in cluster 2 348

were pastured on grass, fed hay and biopsied gastroscopically after a 12-h fast. It is important to 349

note that these two groups of horses had many other differences such as housing, socialization, 350

exercise, and more that may have contributed to the differences in the bacterial communities present 351

(see Table 1 for an overview). De Fombelle et al. (15) commented that the stomach ecosystem 352

seemed the most affected by the composition of the last pelleted meal ingested--hence nutrition and 353

the 12-h fast maybe the main reasons for the differences seen between these two groups of horses. 354

The 12-h fast needed to visualize the stomach by endoscopy may alter bacterial types and numbers 355

because of a lack of substrate available for microorganisms as well as alterations in environmental 356

conditions, specifically a decrease in gastric pH (4, 39, 45,54). The human stomach microbiota had 357

similar bacterial diversity to our gastroscopically sampled horses and was also studied by taking 358

gastroscopic biopsies after a period of fasting(11). In essence, our two non-fasted horses sampled 359

immediately post-mortem demonstrate a unique data set and bacterial community with a 360

predominance of Firmicutes. Although it is unknown how much the gastric microbiota changes in 361

the short time from euthanasia to retrieval of the stomach biopsies. Whether the Firmicutes seen in 362

these two horses represent the normal equine gastric environment and microbiota better than the 363

four horses sampled endoscopically after a 12-h fast is yet to be determined. Another factor that 364

could have contributed to the difference observed between the two clusters is the fact that the 365


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abundance of lactic acid bacteria can be heavily skewed by differences in PCR conditions and 366

methods of DNA isolation (40). While the primary goal of this study was to determine the 367

microbial diversity in normal and abnormal equine gastric mucosa in healthy horses, the 368

overwhelming difference in the composition of the microbiota of samples in cluster 1 versus cluster 369

2 suggests that other factors that were not accounted for in this study may affect the composition of 370

the microbiota. Future studies should therefore take factors like husbandry, sex and age into 371

account in the study design. 372

373

Certain species of Lactobacillus, that have been previously associated with equine fecal matter, 374

are also present in the gastric mucosa of the horses. Phylogenetic analyses of the most dominant 375

read types found among the different samples showed that the sequences obtained by 16S rRNA 376

bacterial tag encoded pyrosequencing (bTEFAP) were phylogenetically informative enough to 377

identify reads assigned to Lactobacillus spp. to species, while reads assigned to other genera (for 378

instance Moraxella) were not. We found one group of reads (the most dominant read type in the 379

antral and glandular samples of horse 2) to be a sister group of L. jensenii and L. fornicalis, which 380

may represent a taxonomically uncharacterized species. In addition to this group we found reads 381

that clustered with the type strains of L. hayakitensis and L. equigenerosi, two distinct Lactobacillus 382

species that were reported to be the predominant lactobacilli in the intestinal microbiota of healthy 383

thoroughbred horses(41). The observation that these species are also abundant in the gastric mucosa 384

of healthy horses suggests that these species may also form a predominant component of the local 385

microbiota of the gastric mucosa. 386

387

Lactobacillaceae and Steptococcaceae form a distinct association with the gastric mucosa. 388


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FISH delivered additional evidence that at least some part of the microbiota observed by 389

pyrosequencing is part of the local microbiota of the equine gastric mucosa, and does not present an 390

amalgam of transitional bacteria that have entered the gastric environment through a nasal 391

pharyngeal route. Lactobacillus and Streptococcus specific probes showed the majority of the 392

bacteria being tightly adhered to the mucosal surface and to any ulcerated mucosa. Similar 393

observations of closely adhered lactobacilli to the squamous epithelium of the horse stomach were 394

made Yuki et al. (62) They identified these Lactobacilli as L. salivarius, L. crispatus, L. reuteri and 395

L. agilis by 16S rDNA sequence analysis and DNA-DNA hybridization. Yuki et al. (62) further 396

found that these bacteria adhered to horse epithelial cells, but not to rat epithelial cells, suggesting 397

host specificity of these Lactobacillus strains. The Yuki et al. study and the results of our FISH 398

experiments combined with the pyrosequencing results thus suggest that the gastric mucosa is 399

inhabited by highly host specific lactobacilli and streptococci. 400

401

Helicobacter is not an abundant taxon in the microbiota of healthy horses. The microbiota 402

found in abnormal (e.g. ulcerated or eroded) regions of the gastric mucosa did not differ from 403

normal regions, nor could we find any specific groups of bacteria among the abundant groups that 404

were specific for ulcerated regions. An important finding was the absence of Helicobacter 405

sequences in over 53,920 reads from healthy horses. On a whole-horse basis, none of the six 406

healthy horses were carriers of Helicobacter spp., however, we acknowledge that the upper 95% 407

confidence limit on 0/9 would allow for as high as 37% carrier prevalence. There has only been 408

weak historical evidence of Helicobacter infection in association with and without ulcers in 409

horses(14, 16) and Helicobacter spp. was not identified in 36 horses with antral pathology using 410

FISH(28). Taken as a whole, these studies indicate that Helicobacter spp. are probably not a 411


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common asymptomatic inhabitant of the equine stomach and their role in gastric ulceration in the 412

horse is yet to be determined. 413

This study establishes the presence of a rich and diverse mucosa-associated bacterial 414

microbiota in the stomach of healthy horses providing a starting point for research on the function of 415

the native microbiota in the equine gastro-intestinal tract. 416

417

Acknowledgements 418

The authors thank M. Baumgart and S. Janecsko for their assistance with the laboratory work 419

and special thanks to Paula Sharp and Francis Davis for their technical assistance. John Parker 420

was supported by the Cornell Leadership Program and the Wellcome Trust and Rebecca 421

Rosenthal was supported by a Research Apprenticeship in Biological Sciences at Cornell 422

University. This project was funded by the United States Equestrian Federation, Inc. 423

424

425

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591

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Figure Legend 593

594

Figure 1. The equine stomach during gross post-mortem and endoscopic examination 595

followed by a closer investigation with histopathology and FISH. a) Post-mortem 596

examination of H3 with a grade 2 ulcer in the squamous portion of the stomach near the margo 597

plicatus. (b) Endscopic view of the cardiac region (H158) of the stomach with small pin-point 598

ulcers (grade 1) at the junction of the squamous and glandular regions. (c) Gram stain of 599

ulcerated mucosa (H158) shows colonization by Gram positive cocci and rods (40x) 600

(d) FISH with probes directed against bacteria in general (EUB338, 6-FAM) and Streptococcus 601

sp. (STREP, Cy-3) showing dense colonization of the ulcerated mucosa by Streptococcus sp. 602

(bright yellow) and fewer other bacteria (green). DAPI stained nuclei appear blue. Original 603

magnification: 60x. 604

605

Figure 2: Hierarchical cluster analysis of samples of the equine gastric mucosa based on 606

relative abundance of sequence reads on the bacterial genus level. The two main clusters 607

found in this analysis coincide with differences in management (feeding, exercise, housing, etc) 608

and sampling method. Horses sampled immediately post-mortem (H2 and H3) and fed hay 609

formed Cluster 1 whereas horses biopsied endoscopically after a 12-hour fast (H35, H60, H79) 610

and fed hay and grass formed Cluster 2. The exception was horse 86 who appeared in both 611

Clusters. An individual horse had a distinct equine gastric mucosa microbiota without 612

differences between regions. Numbers above the nodes are aproximate unbiased values. Labels 613

indicate horse ID along with stomach region (Sq = squamous, G= glandular, A = antral, U= 614

ulcer, E= erosion, P = superficial parakeratosis). 615


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616

Figure 3. Principal Component Analysis plot of the horse stomach samples (solid dots) 617

based on the relative abundance of bacterial genera per horse. Labels indicate horse ID 618

number and stomach region (SQ = squamous, G= glandular, A = antral, U= ulcer, E= erosion, 619

PK = superficial parakeratosis). Names of the bacterial genera with the highest loadings have 620

been placed in the plot according to their correlation to the first two components. 621

622

Figure 4. Principal Coordinate ordination plot of the samples of the horses based on a 623

Bray-Curtis matrix derived from the relative abundance of bacterial genera per horse. 624

Labels indicate horse ID number and stomach region (SQ = squamous, G= glandular, A = antral, 625

U= ulcer, E= erosion, PK = superficial parakeratosis). 626

627

Figure 5. The top five Phyla found in the gastric mucosa of 6 healthy horses by bTEFAP. 628

The top five phyla represented were Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria and 629

Actinobacteria. Cluster 1 horses (H2 and H3) had less diversity with a predominance of 630

Firmicutes (>82%) whereas horses in Cluster 2 (H35, H60, H79, and H86) had a more diverse 631

population consisting of higher percentages of Proteobacteria and Bacteroidetes, as well as 632

Firmicutes. The horse ID number and stomach region (SQ = squamous, G= glandular, A = 633

antral, U= ulcer, E= erosion, PK = superficial parakeratosis) are along the X-axis. 634

635

Figure 6: FISH shows abundant bacteria in close apposition to the equine gastric mucosa. 636

Histologic sections from the squamous (a,d), glandular (b,e) and ulcerated mucosa (c,f) were 637

examined using labeled oligonucleotide probes directed against bacteria in general (EUB-338: 6-638


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FAM, green) in combination with probes directed against Lactobacillus sp. (a,b,c, Cy-3) or 639

Streptococcus spp. (d,e,f, Cy-3). In healthy squamous mucosa (a, yellow) Lactobacillus spp. and 640

(b, yellow) Streptococcus spp. are in close apposition to the epithelium. In the glandular region, 641

both Lactobacillus spp. (b, yellow) and Streptococcus spp. (e, yellow) colonize the mucosa but 642

appear to be lesser amounts than in the squamous region. Ulcerated mucosa from H3 (c,f) 643

contains an abundant mixed bacterial population that includes Streptococcus spp. (f, yellow), 644

Lactobacillus spp. (c, yellow) and other bacterial species (c,f green). Nuclei are blue (DAPI). 645

Original magnification: 600x. 646

647

Figure 7. Healthy horses generally have an abundance of bacteria (<1000 bacteria/mm2) in 648

all gastric mucosal samples by FISH (n=9). FISH was done with probes directed against 649

bacteria in general (eubacteria), Streptococcus spp. and Lactobacillus spp. A few horses (H2, H3 650

and H35) had outliers as indicated. Horse 3’s grade 2 ulcer had an abundance of bacteria 651

(30,529 bacteria/mm2). All other horses had small pin-point ulcers with total number of bacteria 652

similar to other regions of the stomach. Each symbol represents a horse. There was significantly 653

more Streptococcus spp. in the glandular mucosa than the squamous and antral mucosa 654

(P=0.0098) as indicated by the *. 655

656


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Table 1. Table summarizing signalment, management, sample acquisition and analyses per horse .

Horse ID Datea Age

(y)a Breed a Sex a

Management and

Sample Acquisitionb

Gross Ulcer

Severity Grade

Gross Ulcer Number Score

Histopathology of abnormal

mucosa FISH bTEFAP

Pyrosequencing

H1 5/23/06 NR NR G 1 0 0 - + -

H2 5/23/06 NR NR G 1 0 0 - + +

H3 5/31/06 NR NR G 1 2 1 Ulcer + +

H35 6/14/06 7 TB M 2 1 3 Ulcer + +

H60 6/14/06 14 WB M 2 0 0 - + +

H79 6/18/06 17 TB M 2 1 2 Parakeratosis + +

H86 6/14/06 15 TB M 2 1 2 Erosion + +

H131 1/30/07 9 WB G 2 0 0 - + -

H158 1/30/07 8 TB G 2 1 3 Ulcer + -

aDate = date of sampling, y= years, NR = not recorded, TB = thoroughbred, WB = warm blood, G= gelding, and M = mare, NA = not applicable

b 1: Sampled post-mortem, 5 mm puch biopsy, non-fasted 12 h before sampling, non-pastured, exercise a 30 min turnout/day in dirt paddock, horses were subject to student lameness exams. 2: Sampled antemortem, 2-3 mm gastroscopic biopsy, fasted 12 h before sampling, pastured, not subject to student lameness exams.


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Sample IDaNo. of

reads/sample 3% 5% 3% 5% 3% 5%H2-A 3093 125 105 148 122 147 121H2-G 4150 126 107 166 128 163 120H2-SQ 2079 113 100 132 114 151 131H3-A 1278 94 81 126 120 118 108H3-G 1491 77 62 80 54 74 52H3-SQ 4248 62 46 59 42 57 40H3-U 4345 69 48 81 53 96 71H35-A 1613 122 103 129 113 131 116H35-G 2300 175 140 200 157 204 160H35-SQ 3124 226 174 282 216 287 215H35-U 1783 133 109 148 117 146 120H60-A 712 89 80 77 65 81 64H60-G 1778 156 136 172 149 170 149H60-SQ 3033 192 149 235 179 232 174H79-A 1576 197 159 236 181 247 188H79-G 2441 210 165 253 195 243 194H79-PK 2008 189 155 235 185 230 178H79-SQ 2183 209 161 240 175 239 176H86-A 641 80 70 69 62 67 60H86-E 1611 121 97 136 105 131 101H86-G 963 106 87 110 88 104 93H86-SQ 787 93 78 100 80 101 80a A=antral, G=glandular, SQ=squamous, U=ulcer, PK=parakeratosis, E=erosion

Chao1ACEOTU

Table 2. Number of reads, observed taxonomical units (OTUs) and estimated total number of taxonomical units (ACE and Chao1 estimators) at the 3 and 5 % sequence divergence level.


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Table 3. Most abundant species/phylotype in individual samples. Values indicate the percentage of the total number of reads.

Samplea

Phylogroup/speciesb 2A 2G 2SQ 3A 3G 3SQ 3U 35A 35G 35SQ 35U 60A 60G 60SQ 79A 79G 79SQ 79PK 86A 86G 86SQ 86E

Sistergroup to L. jensenii / fornicalis 19 29

L. hayakitensis 19 20

L. equigenerosi 18

Streptococcus sp. 24

Sarcina sp. 13

Eubacterium sp. 14

Actinobacillus sp. 19 6

Moraxella sp. 9 18 11 6 10 10 8 11 19 22

Acinetobacter sp. 7

Veillonella sp. 10

a Horse ID (numeric value) and gastric region are indicated (A=antral, G= glandular, SQ = squamous, U= ulcer, PK= parakeratosis, E= erosion). b Assignment of the most abundant read type to species or phylotype was done using a phylogenetic approach; reads were aligned to existing 16S alignments of type sequences obtained from the RDP database and phylogenetic similarity was inferred by a maximum likelihood analysis (using RaxML version 7.0.4 [53])


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H86

.SQ

H3.

G

H2.

A

H3.

SQ

H2.

SQ

H3.

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H3.

A

86.A

H35

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H35

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H60

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H60

.G

H60

.SQ

H79

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H79

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H79

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H86

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H86

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9599 9999

100 95 959199 82

82

70 96

98 8591 72

95

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96

Figure 2: Hierarchical cluster analysis of samples of the equine gastric mucosa based on relative abundance sequence reads on the bacterial genus level. The two main clusters found in this analysis coincide with differences in management (feeding, exercise, housing, etc) and sampling method. Horses sampled immediately post-mortem (H2 and H3) and fed hay formed Cluster 1 whereas horses biopsied endoscopically after a 12-hour fast (H35, H60, H79) and fed hay and grass formed Cluster 2. The exception was horse 86 who appeared in both Clusters. Labels indicate horse ID along with stomach region (SQ = squamous, G= glandular, A = antral, U= ulcer, E= erosion, PK = superficial parakeratosis).

Cluster 1 Cluster 2


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-0.8 -0.6 -0.4 -0.2 0.0 0.2

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Comp.1 (explained variance, 0.508)

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00)

Gemella

Actinobacillus

Veillonella

Moraxella

Sarcina

Porphyromonas

Lactobacillus

Streptococcus

Prevotella

H2.A

H2.G

H2.SQ

H3.AH3.G

H3.SQ

H3.U

H35.A

H35.G

H35.SQ

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H60.G

H60.SQ

H79.A

H79.G

H79.SQH79.P

H86.A

H86.G

H86.SQ

H86.E

Figure 3. Principal Component Analysis plot of the horse stomach samples (solid dots) based on the relative abundance of bacterial genera per horse. Labels indicate horse ID number and stomach region (SQ = squamous, G= glandular, A = antral, U= ulcer, E= erosion, PK = super-ficial parakeratosis). Names of the bacterial genera with the highest loadings have been placed in the plot according to their correlation to the �rst two components.


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-0.6 -0.4 -0.2 0.0 0.2 0.4

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H60.GH60.SQ

H79.A

H79.G

H79.SQ

H79.PK

H86.A

H86.G

H86.SQ

H86.E

PCoA ordination

Figure 4. Principal Coordinate ordination plot of the samples of the horses based on a Bray-Curtis matrix derived from the relative abundance of bacterial genera per horse. Labels indicate horse ID number and stomach region (SQ = squamous, G= glandular, A = antral, U= ulcer, E= erosion, PK = superficial parakeratosis).


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0%

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2-A 2-G2-SQ3-A 3-G3-SQ3-U35-A35-G35-SQ35-U60-A60-G60-SQ

79-A79-G79-SQ79-PK86-A86-G86-SQ86-E

ActinobacteriaFusobacteriaProteobacteriaBacteroidetesFirmicutes

H2-AH2-GH2-SQH3

-AH3-GH3-SQH3

-UH35-AH3

5-GH35-S

QH35-UH6

0-AH79-A

H60-S

QH60-G

H79-P

KH79-S

QH79-G H8

6-AH86-GH86-S

QH86-E

Figure 5. The top five Phyla found in the gastric mucosa of 6 healthy horses by bTEFAP clustered based on manage-ment and sample method. The top five Phyla represented were Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria and Actinobacteria. Cluster 1 horses (H2 and H3) had less diversity with a predominance of Firmicutes (>82%) whereas horses in Cluster 2 (H35, H60, H79, and H86) had a more diverse population consisting of higher percentages of Proteobacteria and Bacteroidetes, as well as Firmicutes. The horse ID number and stomach region (SQ= squamous, G= glandular, A = antral, U= ulcer, E= erosion, PK = superficial parakeratosis) are along the X-axis.


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Figure 7. Healthy horses generally have an abundance of bacteria (<1000 bacteria/mm2) in all gastric mucosal samples by FISH (n=9). FISH was done with probes directed against bacteria in general (eubacteria), Streptococcus spp. and Lactobacillus spp. A few horses (H2, H3 and H35) had outliers as indicated. Horse 3’s grade 2 ulcer had an abundance of bacteria (30,529 bacteria/mm2). All other horses had small pin-point ulcers with total number of bacteria similar to other regions of the stomach. Each symbol represents a horse. There was significantly more Streptococcus spp. in the glandular mucosa than the squamous and antral mucosa (P=0.0098) as indicated by the *.


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on August 11, 2019 by guest · 36 mainly Streptococcus spp., Lactobacillus spp. and Sarcina spp . Cluster 2 was more diverse, with 37 predominantly Proteobacteria, Bacteroidetes and