Characterization of gastrointestinal inflammatory cell type in equine glandular gastric disease

Heidi E. Banse Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

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Fabio Del Piero Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA
Louisiana Animal Disease Diagnostic Laboratory, Baton Rouge, LA

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Frank M. Andrews Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

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Nicolas Garcia-Abarca Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

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Tatiane Terumi Negrão Watanabe Antech Diagnostics, Los Angeles, CA

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Abstract

OBJECTIVE

To compare small intestinal inflammation with gastric inflammation in horses with and without equine gastric glandular disease (EGGD), we evaluated endoscopic, macroscopic, and microscopic findings of the glandular stomach and microscopic findings of the small intestine.

ANIMALS

36 horses.

METHODS

Horses underwent endoscopy and were scored for EGGD. After euthanasia, stomachs were collected and macroscopically evaluated. Normal pyloric mucosa, glandular lesions, and small intestinal (duodenum, mid-jejunum, and ileum) samples were collected and processed for microscopic examination. Cellular infiltrate was scored. Immunohistochemistry (CD3, CD20, and Iba-1) was performed on the ventral pylorus and small intestine of horses with mild to moderate lymphoplasmacytic infiltrate. A Spearman’s correlation coefficient was used to evaluate the relationship of EGGD grade with gastric glandular inflammation, and the relationships of cellular infiltrate type and severity among glandular stomach, duodenum, jejunum, and ileum.

RESULTS

Gastrointestinal inflammation was common, with gastric inflammatory infiltrate identified in 92%, duodenal inflammatory infiltrate in 83%, jejunal inflammatory infiltrate in 92%, and ileal inflammatory infiltrate in 92% of horses. Endoscopic evidence of gastric disease (hyperemia or EGGD grade ≥ 2/4) was not associated with the presence or severity of duodenal, jejunal, or ileal inflammation. Gastric lymphoplasmacytic inflammation grade ≥ 2 was associated with duodenal lymphoplasmacytic inflammation grade ≥ 2. This was a convenience sample of horses presenting for euthanasia. Medical history (including deworming history) was unknown.

CLINICAL RELEVANCE

Gastric lymphoplasmacytic inflammation is associated with duodenal lymphoplasmacytic inflammation but not more distal small intestinal inflammation. Intestinal inflammation is not associated with endoscopic findings (hyperemia or EGGD grade ≥ 2/4).

Abstract

OBJECTIVE

To compare small intestinal inflammation with gastric inflammation in horses with and without equine gastric glandular disease (EGGD), we evaluated endoscopic, macroscopic, and microscopic findings of the glandular stomach and microscopic findings of the small intestine.

ANIMALS

36 horses.

METHODS

Horses underwent endoscopy and were scored for EGGD. After euthanasia, stomachs were collected and macroscopically evaluated. Normal pyloric mucosa, glandular lesions, and small intestinal (duodenum, mid-jejunum, and ileum) samples were collected and processed for microscopic examination. Cellular infiltrate was scored. Immunohistochemistry (CD3, CD20, and Iba-1) was performed on the ventral pylorus and small intestine of horses with mild to moderate lymphoplasmacytic infiltrate. A Spearman’s correlation coefficient was used to evaluate the relationship of EGGD grade with gastric glandular inflammation, and the relationships of cellular infiltrate type and severity among glandular stomach, duodenum, jejunum, and ileum.

RESULTS

Gastrointestinal inflammation was common, with gastric inflammatory infiltrate identified in 92%, duodenal inflammatory infiltrate in 83%, jejunal inflammatory infiltrate in 92%, and ileal inflammatory infiltrate in 92% of horses. Endoscopic evidence of gastric disease (hyperemia or EGGD grade ≥ 2/4) was not associated with the presence or severity of duodenal, jejunal, or ileal inflammation. Gastric lymphoplasmacytic inflammation grade ≥ 2 was associated with duodenal lymphoplasmacytic inflammation grade ≥ 2. This was a convenience sample of horses presenting for euthanasia. Medical history (including deworming history) was unknown.

CLINICAL RELEVANCE

Gastric lymphoplasmacytic inflammation is associated with duodenal lymphoplasmacytic inflammation but not more distal small intestinal inflammation. Intestinal inflammation is not associated with endoscopic findings (hyperemia or EGGD grade ≥ 2/4).

Equine gastric ulcer syndrome (EGUS) is a common condition in horses that has been associated with clinical signs including colic and inappetence. Recently, EGUS has been further differentiated into equine squamous gastric disease (ESGD) or equine gastric glandular disease (EGGD) based upon the recognition that the etiology of the 2 disorders may differ.1 To date, little is known about mechanisms contributing to the formation of EGGD.

Factors that have been proposed to contribute to EGGD include a breakdown in mucosal defense or an increase in aggressive or triggering factors.2 The key factor implicated in mucosal defense is prostaglandins. Although the role of prostaglandins in naturally occurring diseases has not been evaluated, NSAID-induced models have not shown that a decrease in prostaglandins is associated with EGGD.3,4 Thus, it seems unlikely that a decrease in prostaglandins plays a central role in the development of spontaneous EGGD. Aggressive or triggering factors that have been proposed include stress, bacteria, or inflammation.58 In humans, Helicobacter-negative gastritis has been associated with inflammatory bowel disease.9,10 Furthermore, in dogs, Helicobacter-negative lymphoplasmacytic gastritis has been proposed to be an extension of inflammatory bowel disease (IBD).11 The relationship between inflammatory infiltrates and endoscopic and histopathologic findings of glandular gastric disease in horses remains unexplored. We hypothesized that grade 1 (hyperemic) glandular lesions are associated with lymphoplasmacytic gastritis, while endoscopic grade ≥ 2 (fibrinosuppurative or hyperemic) lesions are associated with erosion or ulceration and neutrophilic infiltration. Furthermore, we hypothesized that gastric inflammatory infiltrate is associated with small intestinal inflammatory infiltrate.

The specific aims of this study were (1) to characterize inflammatory cell infiltrate in horses with and without endoscopic evidence of EGGD; (2) to assess relationships between endoscopic lesion appearance, and macroscopic and microscopic findings; and (3) to evaluate the relationship between inflammatory cell infiltrate in the stomach and small intestine.

Methods

This study was approved by the Louisiana State University Institutional Animal Care and Use Committee (LSU IACUC #18-053). For this study, a power calculation was performed. In a previous study, inflammatory cell infiltrate score (0 = none, mild = 1, moderate = 2, severe = 3) was evaluated and found to be (mean ± SD) 0.9 ± 0.6 in horses without evidence of gross lesions and 1.4 ± 0.6 in horses with lesions.8 Using a β = 0.80 and α = 0.05, it was estimated that 28 horses would be needed to detect differences in inflammatory infiltrate score. Additional horses were included due to potential differences in lesion severity or phenotype in the present cohort.

Endoscopy

Included horses were donated for research. Horses were sedated with xylazine (0.4 mg/kg, IV) after a 16–18 hour period of starvation, and gastroscopy was performed using a 3-m endoscope (Karl Storz, Inc). Lesions (EGGD) were scored using an available scoring system12 by investigators (F.M.A. and H.E.B.). Horses were euthanized, and stomachs were removed and rinsed with water. Stomachs were evaluated and lesion size, appearance, and number were recorded. Since the pylorus is the area that is most commonly affected by EGGD13, samples were collected from normal-appearing portions of the pylorus (2 cm2 from both dorsal and ventral pylorus), as previously described.8 In addition, any area of the glandular mucosa with lesions and a sample of the proximal duodenum, mid-jejunum, and ileum were collected and processed as described for normal-appearing mucosa.

Histopathology

Samples were fixed in 10% neutral-buffered formalin for 48–72 hours, trimmed, dehydrated in progressive alcoholic solutions, paraffin-embedded, sectioned, and stained with hematoxylin and eosin for histopathologic evaluation. Inflammation severity was scored using a previously described 0–3 severity scoring system for small animals.11,14 Additional findings, including the presence of erosion or ulcer, edema, congestion, and intimal asteroid bodies were recorded. Small intestinal sections (duodenum, jejunum, and ileum) were evaluated and scored for inflammation and any other intestinal pathology. Histopathologic evaluation of the samples was performed by 2 pathologists (F.D.P. and T.W.) that were blinded to the endoscopic appearance and clinical scoring.

Immunohistochemistry

Among horses with glandular gastric lymphocytic infiltrate of the ventral pylorus (n = 13 mild and n = 5 moderate), immunohistochemical staining of formalin-fixed samples was used to evaluate T-cell lymphocytes (CD3), B-cell lymphocytes (CD20), and macrophage populations (Iba-1) using methods and antibodies previously validated in horses.1519

In brief, samples from the ventral pylorus were placed in 10 mM citrate buffer, pH 6, for heat-induced antigen retrieval. Samples were blocked with 3% hydrogen peroxide, rinsed, and blocked with goat serum. A 1:400 dilution CD3 (Agilent) or CD20 (ThermoFisher) polyclonal antibody was applied. After rinsing, a biotinylated IgG (Vector Laboratories) was applied. Samples were detected with avidin/biotin complex (Vector Laboratories) and rinsed. An isotype-matched antibody was used as a negative control. Samples were stained with Nova Red (Vector Laboratories) and counter-stained with hematoxylin. All rinsing steps were undertaken with 0.05% TBS-Tween. The number of cells expressing CD3 or CD20 was averaged over 10 fields randomly chosen in high-power magnification (2.37 mm2). Immunohistochemistry was performed in the duodenal, jejunal, and ileal samples as described above, to evaluate the relationship between lymphocyte subtypes between the glandular gastric mucosa and small intestinal mucosa.

The same immunohistochemical staining of formalin-fixed samples was used to evaluate macrophage populations using methods and antibodies previously validated for use in the horse. In brief, samples from the ventral pylorus were placed in 10 mM citrate buffer, pH 6, for heat-induced antigen retrieval. Samples were blocked with 3% hydrogen peroxide, rinsed, and blocked with goat serum. A 1:400 dilution of anti-Iba-1 antibody (FujiFilm Wako Antibodies) was applied. After rinsing, a biotinylated IgG (Vector Laboratories) was applied. Samples were detected with avidin/biotin complex (Vector Laboratories) and rinsed. An isotype-matched antibody was used as a negative control. Samples were stained with Nova Red (Vector Laboratories) and counter-stained with hematoxylin. All rinsing steps were undertaken with 0.05% TBS-Tween. The number of cells expressing Iba-1 was averaged over 10 randomly chosen 40X power fields. Samples of the duodenum, jejunum, and ileum from the horses underwent Iba-1 immunohistochemistry as described above, to evaluate the relationship between lymphocyte subtypes between the glandular gastric mucosa and small intestinal mucosa.

Statistical analysis

Statistical analysis was performed using Prism 9 (GraphPad Software). The type and severity of inflammation in glandular mucosa and small intestinal (duodenal, jejunal, and ileal) mucosa were described based on identified cell type. Number of horses with other microscopic abnormalities of the glandular gastric or small intestinal mucosal mucosa (mucosal edema, congestion, or hemorrhage; plant granulomas and parasitism, or intimal asteroid bodies) were described. Relationships between the severity of microscopic findings and the severity of EGGD were evaluated using Spearman’s correlation coefficient. The severity of glandular inflammation between horses with and without EGGD was compared with a Mann-Whitney U test. Relationships between gastric and small intestinal (duodenal, jejunal, and ileal) inflammation were assessed using Spearman’s correlation coefficient. Numbers of CD3 positive, CD20 positive, and Iba-1 positive cells in the ventral pylorus were compared between horses with mild and moderate lymphoplasmacytic inflammation using a Mann-Whitney U test. Relationships between CD3 positive, CD20 positive, and Iba-1 positive cells in the ventral pylorus and small intestine (duodenum, jejunum, and ileum) were evaluated in horses with mild to moderate lymphoplasmacytic inflammation using a Spearman’s correlation coefficient.

Results

Horses

Thirty-six horses donated to the research herd were included. There were 18 mares, 17 geldings, and 1 stallion. Breeds represented included 23 Thoroughbreds, 7 Quarter Horses, 2 Tennessee Walking Horses, 2 Appaloosas, 1 Warmblood, and 1 Paint. Age was 12 ± 7 years (mean ± SD). Extensive history was not collected. Reported reasons for donation included orthopedic disease (n = 14), old age (n = 5), unknown (n = 5), neurologic disease (n = 4), laminitis (n = 2), pituitary pars intermedia dysfunction (n = 2), presumptive cellulitis (n = 1), increased liver enzymes (n = 1), blindness (n = 1), and cribbing (n = 1).

Endoscopic, macroscopic, and microscopic findings

Postmortem (macroscopic) findings for 1 horse were not recorded. Twenty-three horses had endoscopic hyperemia (Figure 1) and 19 had macroscopic hyperemia. Endoscopic evidence of hyperemia was not associated with macroscopic postmortem evidence of hyperemia (r = 0.3, P = .08), or microscopic evidence of congestion or hemorrhage (r = 0.21, P = .22). Eleven horses had EGGD ≥ 2, 9 had macroscopic ulceration, and 7 had microscopic ulceration. EGGD ≥ 2 (Figure 2) was related to macroscopic (r = 0.62, P = .007) ulceration and microscopic neutrophilic infiltrate (r = 0.55, P < .0001) but not microscopic (r = 0.30, P = .08) ulceration. Microscopic evidence of an ulcer was related to neutrophilic inflammation (r = 0.66, P = .002).

Figure 1
Figure 1

Endoscopic hyperemia.

Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.06.0129

Figure 2
Figure 2
Figure 2

(a) Endoscopic equine gastric glandular disease (EGGD) 2. (b) Microscopic image of an ulcer from a horse with EGGD. Segmental V-shaped necrosis of the gastric mucosa with numerous neutrophils and marginal hyperemia and hemorrhage. Hematoxylin and eosin, 10X.

Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.06.0129

Gastrointestinal inflammation was common, with gastric inflammatory infiltrate identified in 92%, duodenal inflammatory infiltrate in 83%, jejunal inflammatory infiltrate in 92%, and ileal inflammatory infiltrate in 92% of horses (Table 1). Lymphoplasmacytic infiltrate was the most common infiltrate observed in the stomach and duodenum; eosinophils were the most frequently observed cell type in the jejunum and ileum. Eighty-six percent of horses with gastric lymphoplasmacytic infiltrate had duodenal lymphoplasmacytic infiltrate. Other findings within the glandular stomach included intimal asteroid bodies (n = 22), congestion (n = 9), plant granulomas (n = 6), hemorrhage (n = 2), and nematodes (n = 1). Within the duodenum, other findings included intimal asteroid bodies (n = 19), congestion (n = 5), glandular mineralization (n = 4), coccidiosis (n = 1), and congestion with hemorrhage (n = 1). Other findings within the jejunum included congestion (n = 3), coccidiosis (n = 2), and edema (n = 1). Within the ileum, other findings included congestion with hemorrhage (n = 2), hemorrhage (n = 4), edema with hemorrhage (n = 1), coccidiosis (n = 1), and intestinal lymphoma (ileum; n = 1).

Table 1

Region of the gastrointestinal tract, severity of inflammation, and type of inflammatory cell infiltrate.

Region Inflammation (any) (n = 36) Lymphocytes or plasma cells (n = 36) Eosinophils (n = 36) Neutrophils (n = 36)
Mild Mod Sev Tot Mild Mod Sev Tot Mild Mod Sev Tot Mild Mod Sev Tot
Glandular gastric 16 13 4 33 23 9 1 33 9 1 2 13 9 5 1 15
Duodenum 25 5 0 30 25 5 0 30 8 0 0 8 2 0 0 2
Jejunum 25 8 0 33 23 4 0 27 30 3 0 33 2 0 0 2
Ileum 18 13 2 33 22 6 0 28 21 8 0 29 4 0 0 4

The severity of gastric glandular inflammation was not correlated with endoscopic severity (r = −0.15, P = .39). Severity of gastric glandular inflammation was not worse in horses with vs without EGGD (P = .46) or with EGGD ≥ 2 vs without EGGD (P = .55). There was no relationship between presence of EGGD and lymphoplasmacytic (r = −0.03, P = .88) or eosinophilic gastric inflammation (r = −0.03, P = .88), or between hyperemia and inflammation of any type (P ≥ .74). As hyperemia may be indicative of increased blood flow, relationships between endoscopic hyperemia and postmortem congestion or hemorrhage were also examined. No relationship between endoscopic hyperemia and macroscopic hyperemia (P = .08) or microscopic hyperemia or congestion was found (P ≥ .12). Overall, the presence of gastric glandular inflammation was not related to duodenal (r = 0.21, P = .22), jejunal (r = 0.07, P = .68), or ileal inflammation (r = 0.11, P = .51). When examining specific cell types, gastric glandular eosinophilic inflammation was related to duodenal (r = 0.47, P = .004) but not jejunal or ileal (P ≥ .75) eosinophilic inflammation. Glandular neutrophilic or lymphoplasmacytic inflammation was not related to small intestinal neutrophilic or lymphoplasmacytic inflammation (P ≥ .15). However, glandular lymphoplasmacytic inflammation grade ≥2 was related to duodenal (r = 0.43, P = .01) but not jejunal or ileal lymphoplasmacytic inflammation grade ≥2 (P ≥ .71).

Immunohistochemistry

Immunophenotyping results (n = 18) indicated that CD3 positive cells were more common than CD20 positive cells in the ventral pylorus and all segments of the small intestine (P ≤ .0003) (Figure 3). Compared to horses with mild lymphoplasmacytic infiltrate of the ventral pylorus (n = 13), those horses with moderate (n = 5) lymphoplasmacytic infiltrate had fewer CD3 positive cells (P = .0028) (Figures 4 and 5). There were no differences between horses with mild and moderate lymphoplasmacytic infiltrate in CD20 (P = .35) or lba-1 (P = .29) positive cells. Among the 18 horses with mild to moderate lymphoplasmacytic infiltrate of the ventral pylorus, there was no relationship between CD3 positive cells in the ventral pylorus and duodenum (P = .17), jejunum (P = .33), or ileum (P = .11). There was no relationship between CD20 (P ≥ .18) or 1ba-1 (P ≥ .37) positive cells in the ventral pylorus and SI.

Figure 3
Figure 3

Immunohistochemical staining of cells in the ventral pylorus (grey triangles), duodenum (black circles), jejunum (grey circles), and ileum (black triangles) of horses with mild (grade 1) to moderate (grade 2) lymphoplasmacytic infiltration. Open = CD3; filled = CD20. Line = median. CD3 positive cells were more frequent than CD20 positive cells in the ventral pylorus (P < .0001), duodenum (P = .0003), jejunum (P < .0001), and ileum (P = .0002).

Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.06.0129

Figure 4
Figure 4

Immunohistochemical staining of cells in the ventral pylorus of horses with mild (grade 1) vs moderate (grade 2) lymphoplasmacytic infiltration. Number of CD20 positive (dark grey), CD3 positive (black), and Iba-1 positive (light grey) (line = median). Horses with grade 2 lymphoplasmacytic inflammation had fewer CD3 positive cells than horses with grade 1 lymphoplasmacytic inflammation (P = .0028).

Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.06.0129

Figure 5
Figure 5
Figure 5
Figure 5
Figure 5
Figure 5
Figure 5

Representative histopathology microphotographs from horses with mild (grade 1) vs moderate (grade 2) lymphoplasmacytic infiltration in the lamina propria. Horse with grade 1 pyloric inflammation, (a) hematoxylin and eosin (10X), (b) CD20 positive lymphocytes (10X; black arrow), and (c) CD3 positive lymphocytes (10X; black arrow). Horse with grade 2 pyloric inflammation, (d) hematoxylin and eosin (10X), (e) CD20 positive lymphocytes (10X; black arrow), and (f) CD3 positive lymphocytes (10X; none identified in this image).

Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.06.0129

Discussion

In the present study, endoscopic findings of EGGD ≥ 2 were associated with macroscopic findings of ulceration and neutrophilic inflammation, but not microscopic ulceration. There were no other associations between endoscopic findings and microscopic findings. Parallels existed between gastric glandular and duodenal eosinophilic and lymphoplasmacytic (grade ≥2) inflammation, but not between gastric glandular and jejunal or ileal inflammation. These findings suggest that endoscopy is related to postmortem findings of ulceration but may not be helpful in characterizing gastritis type or severity. The relationships between gastric glandular and duodenal inflammation suggest a regionally extensive inflammatory process.

When evaluating the relationship between endoscopic, macroscopic, and microscopic findings, EGGD ≥ 2 was related to macroscopic postmortem identification of ulceration and neutrophilic glandular inflammation, but not microscopic ulceration. Although postmortem findings were related to endoscopic findings, there were some cases where a lesion was identified with endoscopy and not postmortem (likely due to lesion size) or where a lesion was identified postmortem and not on endoscopy (likely due to residual fluid in the stomach at the time of gastroscopy). The lack of correlation between microscopic findings of an ulcer and EGGD ≥ 2 may in part be due to small lesion size, which may not have been visible after fixation in formalin, at the time specimens were cut in for microscopic evaluation. There were no other relationships between endoscopic and microscopic severity of gastric inflammation. In humans, endoscopic assessment of gastritis has also been shown to be poorly related to histologic findings.20 This may be because the endoscopic grading systems incorporate a number or regional extension of lesions12 that cannot be evaluated in a single section of a formalin-fixed sample. Hyperemia (EGGD 1) was not associated with the presence or severity of inflammation (lymphoplasmacytic or other type). Hyperemia is a nonspecific sign of increased blood flow, and it may be that other factors contribute to increased blood flow, rather than inflammation. However, there was a lack of consistent relationship between endoscopic and postmortem evidence of hyperemia, perhaps because congestion occurred during the transport of horses to the necropsy floor. Taken together, these results highlight the limitations of translating endoscopic to pathologic findings.

Asteroid bodies were commonly detected in the glandular gastric and duodenal samples of the horses in this study. Asteroid bodies have been previously identified in multiple equine tissues, most commonly the GI tract, and are considered to be of vascular smooth muscle origin.21 Suspected etiologies include parasitism or trauma.21

Microscopic gastritis was commonly identified in the horses of this study. Two prior studies have identified microscopic gastritis in horses, both with and without endoscopic EGGD.7,8 When examining leukocyte type, lymphoplasmacytic infiltrate was the most commonly identified infiltrate in the glandular gastric mucosa of the present study. Lymphoplasmacytic infiltrate is also the most common infiltrate in dogs with gastritis.22 In people, an increase in lymphocytes may be found in both Helicobacter and non-Helicobacter gastritis.23 Etiologies for non-Helicobacter lymphocytic gastritis in people include celiac disease, viruses, NSAIDs, other medications, lymphoma, or idiopathic.23,24

Parallels observed between glandular gastric inflammation and duodenal inflammation included the presence of eosinophilic inflammation and the presence of lymphoplasmacytic inflammation ≥2, which is likely a reflection of the proximity of the biopsied locations (pylorus and proximal duodenum). In the present study, a nematode was identified in the stomach of 1 horse, and 2 other horses had coccidia identified in the small intestine. Eosinophils are associated with immune disorders, such as allergy or parasitism.25 However, intestinal eosinophils have not always been demonstrated to be related to parasite load,26 and there are other important functions of the eosinophil.27 The scoring system for eosinophil infiltration used was validated in dogs,11 and studies in horses have evaluated inflammation using different measurement methodologies.25,28 Lymphocytes are an important mediator of gastrointestinal immunity and inflammation.29 In people, Helicobacter-negative gastritis is strongly associated with small intestinal inflammation, suggesting an immune-mediated component.9,10 Helicobacter-negative lymphoplasmacytic gastritis in dogs has been proposed to be an extension of IBD.11 Taken together, the findings in the present study suggest that the regional gastroduodenitis observed in the present study may be most consistent with exposure to parasitic or allergic pathogens or other immune-mediated processes. However, as no included horse had specific clinical signs of GI disease (eg, colic, weight loss, or diarrhea), the clinical relevance of these findings is unknown.

When examining lymphocyte type in the ventral pylorus and small intestine, there was a larger number of CD3+ cells identified than CD20+ cells. CD3 is associated with the T cell receptor on mature T-cells. Gastritis in Alaskan racing sled dogs has been identified to be predominated by T lymphocytes, and was suggested to be immune-mediated in origin.30 T cells have been previously demonstrated to be the primary infiltrate in the jejunum of horses with equine eosinophilic enteritis and lymphoplasmacytic enteritis; cell types within the stomach were not evaluated in that study.31 However, when comparing horses with grade 2 vs grade 1 lymphoplasmacytic inflammation in the present study, horses with less severe infiltrate had higher numbers of CD3+ cells.

There are some limitations of the present investigation. Due to the nature of trying to compare endoscopic findings on electively euthanized horses, the exact history of horses (eg, deworming history) was not collected, and findings of inflammation within the stomach or small intestine could be associated with medication administration, parasitism, or other underlying diseases, rather than spontaneous EGGD or inflammatory bowel disease. Furthermore, these horses were collected from a single geographic area (Louisiana) and findings may not relate to horses from other regions. Finally, the EGGD score and degree of microscopic inflammation in the horses of this study were relatively mild. Findings may differ with the inclusion of more severe endoscopic disease or a different microscopic scoring system. This study used established guidelines for small animal scoring of microscopic inflammation, 11,14 as guidelines for equine GI inflammation scoring have yet to be established.32

The results of this study suggest endoscopy is not reflective of microscopic findings of gastric inflammation. The relationship between gastric and duodenal eosinophilic and lymphoplasmacytic inflammation ≥2 suggests a regionally extensive inflammation may be present. Additional investigation into T lymphocyte subtypes may allow for the identification of potential mechanisms contributing to gastric inflammation in horses.

Acknowledgments

Thank you to Dr. Mariano Carossino for providing images of histopathological and immunohistochemical findings.

Disclosures

The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.

Funding

This study was supported by a USDA 1433 (NIFA) grant. Salary support for Nicolas Garcia-Abarca was provided by the Equine Health Studies Program at Louisiana State University.

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