Quantification of immune cells in full thickness and mucosal biopsies of the duodenum and rectum in a group of slaughter horses.

Overview

• This study quantified and compared immune cell populations in full-thickness and mucosal biopsies from the duodenum and rectum of healthy horses.
• The goal was to provide baseline reference values for immune cell counts to aid interpretation in gastrointestinal disease diagnosis.

Introduction and Objectives

• Currently, limited data exist regarding immune cells present in the intestinal walls of healthy horses.
• This lack of baseline information makes it difficult to interpret immune changes during gastrointestinal diseases.
• The study aimed to characterize and quantify immune cell populations in two regions of the horse intestine: the duodenum and rectum.
• It also investigated differences between two types of biopsies: full-thickness biopsies (including all intestinal layers) and mucosal biopsies (endoscopic samples primarily of the mucosa).

Methods

• Subjects: Twenty horses, deemed healthy and fit for slaughter, were sampled.
• Timing: Biopsies were collected within 30 minutes post-slaughter to ensure tissue integrity.
• Biopsy techniques:
  • Full-thickness biopsies (FT) were obtained, sampling the entire intestinal wall.
  • Mucosal biopsies were acquired by endoscopic forceps, sampling primarily the mucosal layer.
• Sampling sites: Duodenum and rectum.
• Histological processing:
  • Tissues were stained with hematoxylin and eosin (HE) for general morphology.
  • Immunohistochemistry (IHC) was used to specifically identify types of immune cells (e.g., B and T lymphocytes, plasma cells).
• Immune cells were evaluated in three anatomical sub-regions:
  • Epithelium
  • Lamina propria
  • Brunner’s glands (duodenum) or submucosa (rectum)
• Statistical analysis:
  • A generalized linear mixed effects model was applied to assess differences in immune cell counts by location and biopsy type.

Results

• Intraepithelial lymphocytes:
  • Count was significantly higher in duodenal full-thickness biopsies (median 9.0 lymphocytes per 100 epithelial cells) than in duodenal mucosal biopsies (median 5.35 per 100 cells).
  • This suggests the full-thickness samples contain more immune cells in the epithelial layer.
• Lamina propria lymphocytes:
  • The duodenal full-thickness biopsies had higher median lymphocyte counts (7 cells/0.01 mm²) than mucosal biopsies (5 cells/0.01 mm²), statistically significant (p < 0.001).
• Plasma cells in lamina propria:
  • Higher counts were observed in duodenal full-thickness biopsies (median 8 cells/0.01 mm²) compared to rectal biopsies, both full-thickness and mucosal (median 4 and 3 cells/0.01 mm², respectively).
• Immune cell identification:
  • The combined B- and T-cell counts via IHC in the duodenal lamina propria were higher than lymphocyte counts identified solely by HE staining (p < 0.001), suggesting IHC is more sensitive and accurate for immune cell detection.

Conclusions and Implications

• Immune cell counts vary significantly depending on:
  • Intestinal location (duodenum vs. rectum)
  • Type of biopsy (full-thickness vs. mucosal)
• Therefore, establishing separate reference values for each biopsy type and location is important for proper interpretation in clinical and research settings.
• Immunohistochemistry enhances accurate identification and quantification of immune cell populations compared to traditional HE staining alone.
• The availability of these baseline reference data will help veterinarians and researchers better understand immune responses in equine gastrointestinal diseases.