Examination of equine glandular stomach lesions for bacteria, including Helicobacter spp by fluorescence in situ hybridisation

Abstract

Background: The equine glandular stomach is commonly affected by erosion and ulceration. The aim of this study was to assess whether bacteria, including Helicobacter, could be involved in the aetiology of gastric glandular lesions seen in horses.

Results: Stomach lesions, as well as normal appearing mucosa were obtained from horses slaughtered for human consumption. All samples were tested for urease activity using the Pyloritek assay, while mucosal bacterial content was evaluated using Fluorescence In Situ Hybridisation. In selected sub samples, bacteria characterisation was pursued further by cloning and sequencing. Mucosal lesions were found in 36/63 stomachs and included hyperplastic rugae, polypoid structures and focal erosions. None of the samples were tested positive for urease activity or for FISH using the Helicobacter genus specific probe. In samples of lesions, as well as normal samples, clones with 99% similarities to Lactobacillus salivarius and Sarcina ventriculi were found. Escherichia like bacterium clones and Enterococcus clones were demonstrated in one focal erosion. Based on a phylogenetic tree these clones had 100% similarity to Escherichia fergusonii and Enterococcus faecium. The Enterococcus were found colonising the mucosal surface, while E. fergusonii organisms were also demonstrated intraepithelial.

Conclusion: Gastric Helicobacter spp. could not be verified as being involved in lesions of the glandular stomach of the horse. Since E. fergusonii has been described as an emerging pathogen in both humans and animals, the finding of this bacterium in gastric erosion warrants further clarification to whether gastric infection with this type bacterium is important for horses.

Figure 1

Focal erosive lesion (white arrow) demonstrating bacterial gastritis at histological evaluation. Lesion was approximately 2 × 2 cm and located in the antrum near the pyloric entrance.

Figure 2

Gastric mucosa with erosive gastritis associated with bacteria. The mucosal surface and adjacent cellular debris is severely colonised by bacteria (red). A few bacteria are seen intracellular in the intact epithelium (arrowhead) as well as within degenerated and necrotic epithelial cells (arrow). In addition, bacteria are found within granulocytes. Fluorescent in situ hybridisation with the probe targeting Bacteria, filter set 43, bar = 25 μm.

Figure 3

A phylogenetic tree of 16S rRNA gene sequence similarity, showing the position of the six clones belonging to Gammaproteobacteria found in Horse 50L and the most closely related type strains belonging to the Escherichia genus. The six clones (acc.no. GQ423062) had 100% similarity to Shigella flexneri and E. fergusonii. Enterobacter sakazakii (AB004746) was used as an outgroup. Sequence accession numbers are presented.

Figure 4

Gastric mucosa of horse 50L with erosive gastritis associated with bacteria. Applying a fluorescein labelled probe for Gammaproteobacteria and a Cy3 labelled probe for Enterococcus, an E. coli like organism (green) (arrowhead) was found intracellular within epithelial cells and on the epithelial surface whereas E. faecium (red) ('white star'(only colonised the epithelial surface. Filter set 43/38, bar = 10 μm.

Figure 5

Gastric mucosa of horse 50L with erosive gastritis associated with bacteria. High magnification demonstrating E. coli like rods (green) within extruded epithelial cells. Fluorescent in situ hybridisation with the probe targeting Gammaproteobacteria, filter set 38, bar = 10 μm.

Figure 6

Anatomical regions of the stomach opened along the greater curvature. The non-glandular region has a white appearing epithelium, whereas the glandular region is shades of red. They are separated by the Margo plicatus. The three sampled regions include: Cardia as the small strip area just below and along the margo plicatus, the corpus region containing acid, pepsinogen and mucus secreting glands (dark red) and the antrum region containing primarly mucus and gastrin secreting glands.