title: Investigation of the equine glandular gastric microbiota in association with equine glandular gastric disease in thoroughbred racehorses using 16S sequencing
Creators: name: Voss, Sarah J.
Publisher: name: University of Glasgow
Year: 2020

description: Background: Equine glandular gastric disease (EGGD) is a condition common to sports and leisure horses world wide, and is characterised by inflammatory and erosive lesions affecting the glandular gastric mucosa. The aetiopathogenesis of EGGD has not been fully elucidated, and success rates of treatment are poor compared to equine squamous gastric disease (ESGD). The role of pathogenic bacteria has often been proposed, but no single pathogen has been consistently identified in association with EGGD lesions. Despite altered gastrointestinal microbiota community profiles being implicated in many other disease processes in the horse, the normal equine gastric microbiota has not been established. Establishing the normal and EGGD-associated glandular gastric microbiota may help to improve understanding of the aetiopathogenesis of this common but poorly understood syndrome, and may assist in optimising treatment protocols.

Objectives: Investigate the equine gastric glandular microbiota in vivo in a population of 
 Thoroughbred racehorses subject to the same management conditions. We aimed to evaluate the microbial population at sites of EGGD lesions compared to normal mucosa, and investigate whether a dysbiosis or specific candidate pathogen may be implicated in the pathogenesis of EGGD. A secondary objective was to explore the use of transendoscopic cytology brushes for acquisition of mucosal samples appropriate for 16S sequencing.

Hypothesis: We hypothesised that the glandular gastric microbiota would show inter individual variation, but that a dysbiosis would be associated with EGGD lesions in a population of Thoroughbred racehorses.

Materials and Methods: Two different cohorts of Thoroughbred racehorses were examined (Yard One, and Yard Two). At Yard One, eight Thoroughbred racehorses from one racing yard were examined gastroscopically following a report of poor performance from the trainer. Samples were taken from EGGD lesions and adjacent normal mucosa using guarded transendoscopic cytology brushes and frozen at -80 °C. DNA was extracted for 16S rRNA sequencing, and sequences compared against a database to generate taxonomic classification of the microbiota. At Yard Two, five Thoroughbred racehorses from one training centre underwent gastroscopy as part of poor performance investigation The same horses, and one additional animal, were sampled again six months later. Samples underwent a similar DNA extraction and sequencing process as for Yard One.

Results: In Yard One samples Proteobacteria was the predominant phylum present (median abundance of 67% at normal mucosa, and 65% at EGGD lesions). This was followed by Bacteroidetes and Firmicutes. Alpha diversity analysis demonstrated EGGD lesions to be more diverse compared to normal mucosa; a finding that approached statistical significance (p = 0.052). Yard Two normal mucosa samples were characterised by a higher proportion of Proteobacteria (46.3 %) compared to lesions (18.9 %) (p = 0.017). Relative abundance of Firmicutes was lower in samples from normal mucosa (20.0 %) compared to lesions (41.2 %) (p = 0.006). Linear discriminant analysis effect size (LEfSe) confirmed that a greater proportion of Firmicutes species was characteristic of samples collected from EGGD lesions. This was due to a very high relative abundance of Sarcina (up to 92.4 %) associated with EGGD lesions in two horses from Yard Two. Across both data sets weighted and unweighted UniFrac analysis demonstrated that samples were of increased similarity if they were collected from the same horse, or collected from the same yard. This effect was stronger than whether samples were from EGGD lesions or normal mucosa. Finally, samples were pooled and recategorized into three groups according to lesion description. When beta diversity analysis was performed on the pooled sample samples did not appear similar according to which lesion group they represented. 

Conclusion: There is evidence to suggest that the gastric microbiome is altered in horses with EGGD, although we are unable to demonstrate a causative effect. Sarcina was identified as a potential biomarker of disease and warrants further investigation as a gastric pathogen in the horse. There is an effect of interindividual variation on the gastric microbiota community, and this effect appears to be of more importance than the difference between normal and diseased glandular mucosa. We have also demonstrated that sheathed cytology brushes can be used as an effective method of sampling the gastrointestinal 
microbiota transendoscopically in live animals.