Gastrine sérique et entérite chronique lymphocytaire-plasmocytaire du chien. Le but de cette étude était d’évaluer les concentrations sériques de gastrine chez des chiens atteins d’entérite lymphocytaire-plasmocytaire chronique ainsi que les éventuelles relations avec la sévérité des lésions présentes dans l’estomac. Dans ce but, 5 chiens témoins sans maladie gastro-intestinale et 15 chiens malades atteints d’entérite lymphocytaire-plasmocytaire chronique ont été utilisés. Les concentrations de gastrine sérique étaient significativement augmentées chez les malades en comparaison des témoins. Il y avait également une corrélation positive entre la sévérité des lésions gastriques et la concentration de gastrine sérique. Nos résultats indiquent la possibilité d’une implication de la gastrine dans l’étiologie de la gastrite chronique de l’antre du pylore qui accompagne l’entérite lymphocytaire-plasmocytaire chronique canine.

(Traduit par Docteur André Blouin)

Chronic lymphocytic-plasmacytic enteritis is an idiopathic chronic gastrointestinal disorder characterized by a diffuse infiltration of lymphocytes and plasma cells in the lamina propria of the small bowel. It is one of a group of canine intestinal diseases termed inflammatory bowel disease (IBD), considered to be the most common cause of chronic vomiting and diarrhea in dogs (1–3).

The definitive cause of this disease remains unknown. Classically, the most commonly hypothesized are immune-mediated, parasitic, and dietary causes (1,4,5). Nowadays, interactions among the mucosal immune system, host genetic susceptibility, and environmental factors are implicated as causing intestinal inflammation. Intestinal inflammation is either due to an abnormal immune response (defective suppressor function of gut associated lymphoid tissue [GALT], host hypersensitivity caused by increased intestinal permeability, other primary immunological events, or all of these causes) or initiated by an appropriate luminal constituent (dietary antigen or resident enteric flora). Two mechanisms have been proposed to explain the ill-defined pathogenesis: An abnormal intestinal permeability and a defective immunoregulation of GALT (1).

Vomiting and diarrhea are the clinical signs more commonly seen in dogs with chronic lymphocytic-plasmacytic enteritis, sometimes associated with weight loss, appetite disorders, and bouts of abdominal pain. The vomitus usually consists of bile without food. The mechanism of vomiting in these dogs is thought to be due to the presence of a chronic antral gastritis, secondary to duodenogastric reflux and pyloric incompetence, possibly provoked by the small bowel inflammation (6,7). The diarrhea is chronic, usually with the typical signs of small intestinal diarrhea.

A diagnosis of chronic lymphocytic-plasmacytic enteritis is one of exclusion and requires the ruling out of many other diseases that may cause intestinal inflammation (8). Once a diagnostic exclusion protocol has been carried out, upper gastrointestinal endoscopy with intestinal biopsy is the diagnostic test of choice. However, this technique presents limitations and, at times, full thickness biopsies of the intestines are required, especially to rule out gastrointestinal lymphoma.

The presence of gastric lesions in dogs with chronic lympocytic-plasmacytic enteritis was the impetus to carry out this study to evaluate a possible relationship between serum gastrin concentrations and gastric lesions.

Gastrin is synthesized and secreted mainly by gastrin (G) cells located in the mucosa of the gastric antrum. Stimulation of these endocrine cells leads to gastrin being released into the circulation. Three major biologically active molecular forms of gastrin exist, as follows: big gastrin or G-34 (formed by 34 amino acids), little gastrin or G-17 (17 amino acids), and minigastrin or G-14 (14 amino acids). This hormone, whose major physiological effect is the stimulation of gastric acid secretion, seems to play an important role in all gastric inflammatory processes (9). For these reasons, serum gastrin evaluation could help in the appraisal of the gastric damage that can appear in chronic lymphocytic-plasmacytic enteritis.

Twenty dogs (11 males and 9 females), 2 to 8 y of age, were included in this study. Animals were divided into 2 groups: Group A — dogs without gastrointestinal disease (n = 5), and group B — dogs with chronic lymphocytic-plasmacytic enteritis (n = 15). Dogs in group A were symptom free and came from owners who voluntarily consented to collaborate in the study. Dogs in group B had gastrointestinal signs (Table 1). All dogs from both groups arrived between January and May 2003 at the Veterinary Medicine Teaching Hospital (VMTH) of the University of Madrid.

Table 1 Clinical signs of dogs included in this study (group A — dogs without gastrointestinal disease, and group B — dogs with chronic lymphocytic-plasmacytic enteritis)

All the animals were subjected to a diagnostic evaluation protocol with a view to their inclusion in group A or B. This protocol included a complete anamnesis, an evaluation of clinical signs, a physical examination, a hematological and biochemical profile, fecal examination for 3 consecutive days for cestodes, nematodes, and protozoa (direct smear with saline solution [10], direct smear with methiolate iodine formaldehyde [MIF] solution [10], and zinc-sulfate flotation or Telemann technique [10]), fecal chymotrypsin and serum trypsin-like immunoreactivity (serum TLI) (Faculty of Veterinary Medicine, Utrecht, Holland), and finally a gastroduodenoscopy in order to obtain biopsy specimens.

Food was withheld for 24 h and water was withheld for 12 h before commencement of the endoscopy. During endoscopic examination (Fujinon EG-200FP videoendoscope, 110 cm long and 9.8 mm in diameter, or Fujinon EC-200LR, 180 cm long and 14 mm in diameter, depending on the size of the animal; Fujinon, [Europe] GmbH, Willich, Germany) of all animals, gross findings were evaluated in the stomach (bile, mucosal erythema, rugal folds, and erosions) and duodenum (mucosal erythema, erosions, granularity, and tissue friability). Based on these gross observations (stomach and duodenum, separately), dogs were classified into 4 categories: no lesion (−), mild changes (+), moderate changes (++), and severe changes (+++). Next, stomach and duodenum were biopsied in all dogs. Biopsies of the stomach were taken from the gastric body (6 samples) and the pyloric antrum (6 samples). Biopsies of the small intestine (8 samples) were taken from different areas of the descending duodenum. Samples were fixed by immersion in neutral-buffered 10% formalin, embedded in paraffin wax, sectioned at 5 mm, and stained with hematoxylin and eosin, Masson trichromic, and periodic acid-Schiff (PAS) techniques. Sections were examined by 1 of the authors (AR), using light microscopy.

The biopsies were evaluated by the Pathology Service of the VMTH. Because there is no world-wide standard for the histological evaluation of the gastrointestinal tract of dogs, we developed our own classification for staging histological findings. Dogs were classified (stomach and duodenum, separately) into 4 categories: no lesion (−), mild changes (+), moderate changes (++), and severe changes (+++); several parameters were evaluated: stomach (epithelial hyperplasia, fibrosis, edema, and infiltration of chronic inflammatory cells) and duodenum (presence of intraepithelium lymphocytes, epithelial continuity, globet cell hyperplasia, grade of infiltration of chronic inflammatory cells, presence of lymphangiectasia, edema, fibrosis, and gland hyperplasia).

Gastric lesions were classified on the basis of the gross appearance of the gastric mucosa and the histopathological lesions in the biopsies into the following categories: absence (in dogs without gastrointestinal disease, group A), moderate and severe (in dogs with chronic lymphocytic-plasmacytic enteritis, group B).

To determine serum gastrin concentrations, 5 mL of blood was collected in glass tubes from each dog immediately prior to endoscopic evaluation, before sedation or the induction of anesthesia. The tubes were immediately centrifuged at 1000 × g for 10 min. Serum was removed and frozen at −5ºC for further analysis. Serum gastrin concentrations were measured by radioimmunoassay, using a commercially available kit (Gastrin J-125 RIA kit; Aurica DRG Diagnostics, DRG Instruments GmbH, Marburg, Germany). The assay is validated for the species, and samples were assayed in duplicate. Mean gastrin concentrations were used in this study.

Briefly, the assay procedure was as follows: 200 μL of gastrin standard (0, 15, 25, 50, 100, 200, 500, and 1000 ng/L) or serum sample was incubated with 100 μL of gastrin tracer solution (Gastrin ¹²⁵J; Aurica DRG Diagnostics) and 100 μL of gastrin antiserum (rabbit anti-human gastrin) for 120 min at room temperature. The 100 μL of tracer was dispensed to only tubes 1 and 2. Afterwards, 1.0 mL of precipitating antiserum was added to all tubes, except 1 and 2, and thoroughly mixed. All tubes, except 1 and 2, were centrifuged at 1500 × g for 15 min. Supernatants were aspirated from all tubes, except 1 and 2, and radioactivity of the precipitates was measured in each tube by counting in a gamma counter for 1 min. Concentrations of gastrin in serum of dogs were determined by interpolation from the standard curve of % trace binding versus ng/L gastrin.

The Wilcoxon test and nonparametric analysis of variance (ANOVA) were used for statistical analysis of the results (statistical program 4.16. Med Calc; MedCalc Software, Mariakerke, Belgium). Significance was considered at P < 0.05.

No abnormalities were observed on the endoscopic exploration or the histological evaluation of the biopsies obtained from group A dogs. On the other hand, in all group B dogs, abnormalities were observed on endoscopic exploration and histological evaluation (Table 2). Gastric lesions located in the pyloric antrum were categorized as follows: absence (5/20), moderate (9/20), and severe (6/20). The duodenal histological lesions were categorized as moderate in all dogs (15/15).

Table 2 Gross endoscopic and histopathological findings (stomach and duodenum) in all dogs included in this study (group A — dogs without gastrointestinal disease, and group B — dogs with chronic lymphocytic-plasmacytic enteritis)

Mean serum gastrin value (standard error of the mean [s]) for the group A dogs was 22.21, s = 3.65 ng/L. Mean serum gastrin value for group B dogs was 40.62, s = 4.36 ng/L. Significant difference between both groups was observed after statistical analysis (Wilcoxon test); serum gastrin values were significantly elevated in group B as compared with group A. These results are summarized in Table 3.

Table 3 Mean serum gastrin values (ng/L) in dogs without gastrointestinal disease (group A) and dogs with chronic lymphocytic-plasmacytic enteritis (group B)

Table 4 Statistical analysis (nonparametric analysis of variance [ANOVA]): serum gastrin concentrations (ng/L) according to gastric lesion degree

Radioimmunoassay is still by far the most reliable tool for the measurement of serum gastrointestinal hormones (11), and the method we used was sufficiently sensitive to measure canine serum gastrin concentrations; the mean serum gastrin concentration for the group A dogs corresponds well with previously reported serum gastrin values (12–14).

The finding of significantly high serum gastrin in chronic lymphocytic-plasmacytic enteritis could be explained by the duodenogastric reflux frequently associated with this disease (6,7). Reflux of duodenal contents (bile, pancreatic juice, and other duodenal secretions) into the stomach would damage the gastric mucosa barrier. This alkaline reflux would also lead to an antral hypomotility and a gastric acid hypersecretion. Our findings suggest that gastrin could play a role in the pathophysiology of the gastric lesions that occur in canine chronic lymphocytic-plasmacytic enteritis.

There are no published reports of serum gastrin concentrations in dogs with chronic lymphocytic-plasmacytic enteritis. Hypergastrinemia has been documented in some Basenji dogs with immunoproliferative enteropathy, although the role of hypergastrinemia in this disease is unclear (15). Immunoproliferative enteropathy of Basenji dogs is a specific entity within chronic lymphocytic-plasmacytic enteritis, exclusively limited to this breed. Nevertheless, our results suggest that hypergastrinemia could exist in other breeds with chronic lymphocytic-plasmacytic enteritis.

Many reports have been published referring to possible associations between human inflammatory bowel disease and changes in serum gastrin levels. Results obtained in these studies differ considerably. Some studies have shown increased concentration of serum gastrin, whereas others have found gastrin values within reference ranges (16,17). However, most reports concerning serum gastrin concentrations in humans with inflammatory bowel disease refer to colonic inflammatory bowel disease.

All dogs with chronic lymphocytic-plasmacytic enteritis included in this study developed moderate to severe lesions located in the pyloric antrum. The discovery of gastric lesions is commonly observed in canine enteritis. We have previously reported that 84 of 100 dogs with chronic lymphocytic-plasmacytic enteritis showed gastric lesions (18).

Dogs with severe gastric lesions had significantly increased serum gastrin values compared with those without gastric lesions, presumably because the higher the number lesions in the stomach, the greater the stimulation of G cells.

Finally, as the results of this study suggest, gastrin is possibly involved in this complex disease, so future research should explore the role of gastrin, including gastric secretory function in dogs with chronic lymphocytic-plasmacytic enteritis.

The higher serum gastrin in dogs with chronic antral gastritis compared with those without gastrointestinal disease, together with the high association observed in our experience between antral gastritis and lymphocytic-plasmacytic enteritis (7,18), is supportive of antisecretory drugs (histamine H₂-receptor antagonists, proton pomp inhibitors) being administered to some dogs with chronic lymphocytic-plasmacytic enteritis. However, additional studies are needed to conclusively clarify some questions still existing about chronic lymphocytic-plasmacytic enteritis in the dog.