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Bulletin UASVM, Veterinary Medicine 65(1)/2008 pISSN 1843-5270; eISSN 1843-5378

MORPHOLOGICAL ASPECTS IN GASTRITIS INDUCED BY Helicobacter pylori IN DOGS

Taulescu M., C. Catoi, A. Gal, P. Bolfa, I. Rus

University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Department of Pathology

3-5 Mănăştur Street, 400372 Cluj-Napoca, Romania taulescumarian@yahoo.com

Keywords: helicobacter pylori; gastritis; histopathology; immunohistochemistry. Abstract: Helicobacter pylori is one of the most common bacterial pathogens in humans. The infection with these bacteria is now recognized as a worldwide problem. It causes chronic gastritis; peptic ulcer disease; and lymphoproliferative disorders and is a major risk factor for gastric cancer. The aim of this research was to investigate the gastric disorders from dogs with spontaneous Helicobacter pylori infection by necropsy and histopathological exams; also identification of the Helicobacter pylori bacteria by immunohistochemistry method in tissues with lesions. At necropsy and histopatological exams we found more specific lesions of the Helicobacter species infection; respectively Helicobacter pylori infection in dogs. The most common types of specific gastritis were chronic follicular gastritis; fibrous gastritis and ulcerative gastritis. The lesions was main present in pyloric and fundic areas. At two cases was not observed lesions. Detection of Helicobacter pylori bacteria has been accomplished by immunohistochemistry method using policlonal antibody.

INTRODUCTION

Spiral gastric bacteria were first seen and described in human stomachs and stomachs of domestic carnivores at the end of the 19th century; but scientific interest in them increased after 1983; when Warren and Marshall (1983) established a relationship between Helicobacter pylori infection and gastritis in humans. The Gram-negative bacterium H. pylori is present in the stomach of approximately 50% ofthe world’s population and is one of the most common causes of chronic gastritis (Robić; M.; 2007). H. pylori infection is associated with the development of chronic gastritis; peptic ulcer disease; gastric adenocarcinoma; and gastric mucosa-associated lymphoid tissue (MALT) lymphoma (Kuipers EJ; 1997). Helicobacters colonize the stomachs and intestines of humans and several animal species; such as cats; dogs; ferrets; pigs; cheetahs; and monkey (Rozmiarek H. et al.; 1994).

All those bacteria in domestic carnivores are referred to as gastric Helicobacter-like organisms (GHLO) and are microaerophilic; gram negative spiral bacteria with numerous flagella and high urease activity; which enables them to survive in an acid environment (Lecoindre et al.; 2000). In dogs all GHLO are of similar length (5-15 µm) and width (0.3-1.2 µm) (Jalava et al.; 1997). Untill now the clinical relevance of GHLO is not known; but it is suspected that those bacteriaare not saprophytic; since in the majority of animals histopathological changes in gastric mucosa; including dilatation of gastric glands; degenerative changes in superficial epithelia and necrosis of individual gastric glands are present. Also; inflammatory changes with increased number of neutrophils and lymphocytes in gastric epithelia. Lymphoid follicles are a common finding (Eaton et al.; 1996). Helicobacter – like organism infection in dog induce more specific lesions; such as chronic gastritis; chronic atrophic gastritis; chronic ulcerative gastritis. This is in accordance with literature data concerning Helicobacter infections in dogs; namely; that those organisms are

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not connected with peptic ulcer disease in domestic carnivora. Chronic erosive gastritis was diagnosed in three animals and chronic atrophic gastritis in four animals (Robić; M.; 2007). Natural infection with H. pylori has been described in cats (Handt et al.; 1994). These H. pylori-infected cats showed lymphoplasmacytic infiltrates with small numbers of PMNs and formation of lymphoid follicles (De Bock; 2006). Natural infection with Helicobacter pylori has been identified in one dog by PCR method in other study (Buczolits; S. et al.; 2003).

Diagnosis of Helicobacter species infection is usually made by histologic examination of endoscopic or postmortem stomach specimens through demonstration of mucosal inflammation accompanied by organisms. Immunohistochemisty; in situ hybridization; electron microscopy and PCR amplification of 16S ribosomal RNA amplicons can be used to differentiate Helicobacter species (Fox J. 2006).

MATERIALS AND METHODS

For this work we used stomach samples from 13 dogs (5 males and 8 females); various breeds; with ages between 2 and 12 years.

At necropsy exam; the stomach samples were harvest from cardial; fundic and piloric regions of the stomach.

Bacterioscopic exam. The smears were made from the three sections of gastric mucosa; using May – Grumwald – Giemsa stain. At the end were identified the germs from the smear and it was made a quantification of the settlement rate with Helicobacter pylori; using the following standard scald : rate 0 (absence of germs); first grade (few germs/smear); second grade (1-5 germs/microscopic field); third grade (more than 5 germs/microscopic field).

For histopathological exam; the stomach samples were preserved in formalin 10% for 36 hours; after that they were cutted at 4-5 mm thickness; and then were automatic processed (brief fixation; dehidratation; paraffin inclusion and embedding). The paraffin blocks were cutted at 3-5 microns thickness and were stained with Hematoxiline-Eozine. The examination was made using Olympus BX51 microscope with digital camera.

The immunohistochemistry method: slides were incubated at 37˚C for 24 h; deparaffination in xilen for 2 hours and 30min.; rehidratation with 96% alcohol three times; rinse with distilled water two times; Tris buffer saline (TBS) for 5 min.; heating at microwave in citric acid solution for 3-5 min.; cooling (20 min.) at room temperature; marking of the sections with Dako pen; Protein Block fot 10 min.;Tris buffer saline for 5 min; additioning of H2O2 for 10 min.; TBS for 5 min; 100 µl of primary antibody Policlonal Rabit anti Helicobacter Pylori (Dako) 1:10 on slide in humid room for 1 hour.; TBS-rinse for three times in Borell glass; 100 µl secondary antibody on slide for 15 min.; TBS- rinse for two times in Borell glass; streptavidine solution for 15 min.; TBS-rinse for two times in Borell glass; Diaminobenzidine diluated solution (DAB) for 2 min.; rinse with distilled water in four Borell glass; counterstaining with hematoxiline for 1 min.; rinse with natural water; rinse in lithium carbonate saturated solution; rinse in water; rinse in 96% alcohol in two Borell glass; rinse in absolute alcohol in one Borell glass; clarification with Xilen for 5 min.; mounting and examination of the slides.

RESULTS AND DISCUSSIONS

At necropsy and histopatological exams we found more specific lesions of the Helicobacter species infection; respectively Helicobacter pylori infection in dogs. Predominant lesions consisted of fibrous gastritis (5 cases); exprimed through the presence of a plentiful fibrous tissue among pits gastric and glands causing their atrophy of compression

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and an infiltrate with mononuclear cells dominated by lymphocytes; macrophage and plasmocytes. In other cases was observed chronic follicular gastritis both in pylor region and fundic region (4 cases); characterized by a lymphocytic follicle reaction in submucosa and lymphocytes infiltrate among pits gastric. Lesions of gastric ulcer were observed at 2 cases; exprimed by PMN infiltrate in the surface layer of the gastric mucosa and epithelial cells necrosis. At 2 cases was observed the absence of macroscopic and histological lesions.

Helicobacter pylori were identified by immunohistochemistry method. They are brown spiral organisms with 3-5/0;5 diameter on the surface layer of gastric epithelium and lumen glands (7 cases). Other six cases were negative for immunohistochemistry stain for Helicobacter pylori. In one sample was observed the presence of Helicobacter felis –like; negative for IHC.

Acute infection with Helicobacter pylori produce acute gastritis characterized by PMN infiltrate in corion of the gastric mucosa and also in surface layer of the epithelium which induce the epithelium necrosis.

The chronic stade is characterized by a mononuclear infiltrate in own lamina; dominated by lymphocytes and plasmocytes and plentiful fibrous tissue in corion that induced the thicken of gastric mucosa.

Major gastric lesions were the following: chronic gastritis with mononuclear cells infiltrate and atrophyc glands; acute stomach ulcers and lymph follicular chronic gastritis.

The lesions were both in pylor and fundic areas.

Cases =13

31%

15%39%

15% Lymph follicular chronic gastritis

Acute stomach ulcer

Chronic gastritis withmononuclear cells infiltrate

Gastric mucosa without lesions

Fig.1 Main gastric lesions found at dogs necropsy

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Photo 1. Chronic follicular gastritis in the pyloric region; HE x100

Photo 2. Chronic follicular gastritis in the fundic region; HE x 200

Photo 3. Stomach ulcer in the pyloric area; epithelial cells necrosis and the neutrophilic infiltrate in the mucosal corion; HE x 100.

Photo 4. Stomach ulcer in the pyloric region; epithelial cells necrosis and the neutrophilic

cells infiltrate in the corion; HE x 100.

Photo 5. Chronic gastritis in the surface layer of the fundic region with fibrosis and mononuclear

cells infiltrate. TM x 200

Photo 6. Chronic gastritis of the pyloric region with fibrosis and mononuclear cells infiltrate;

atrophic glands; TM x 200

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Photo 7. Chronic gastritis of the pyloric region with fibrosis and limphocytes and plasmocytes

infiltrate; positive reaction for Helicobacter pylori (see the red arrow); IHC x 700

Photo 8. Positive reaction for Helicobacter pylori; these is brown marked and characteristic shape

(see the red arrow); IHC x1000

In our study we found a corelation between the Helicobacter pylori gastric infection

grade; gastric lesions severity and Helicobacter pylori positive reaction at IHC. Helicobacter pylori is a pathogen organism for dogs. Acute lesions produced by this

bacteria are similar with the lesions described at other animals as following:acute gastritis with PMN infiltrate; acute stomach ulcer; chronic gastritis (Baba et al.; 1997). Dogs can be infected with Helicobacter pylori; but there are few references about gastric pathology at dogs which describe this infection. In one study made on several dogs; it was diagnosed by PCR method only one case positive for Helicobacter pylori (Buczolits et al.; 2003). Other autors identified this bacteria from feces by analyse of Helicobacter pylori antigen with the specific kits. From a number of 30 dogs; the autors found only one case positive.

Our study proves that at immunohistochemical exam 7 dogs were positive for Helicobacter pylori. At the other cases; the gastric lesions were produced by infection with other species of Helicobacter (H. felis; heilmannii; bizzozeroni) or other causes such as stress; non-steroidal anti-inflammatory drugs (NSAIDs); etc.

At bacterioscopy exam of gastric smears was observed a gastric infection with organisms similar to Helicobacter pylori and Helicobacter felis. The infection with these organisms is second grade that represent an average infection; except one case which is engage at third infection grade. For Helicobacter pylori diagnosis; the immunohistochemistry is very specific and sensitive; low cost and superior to conventional methods (HE şi Giemsa) (Dabbs; 2006). The infection with Helicobacter pylori at dogs and cats can explaine the correlation between this infection at pets and the infection risk at humans; this animals being source of Helicobacter pylori infection for humans.

CONCLUSIONS

� Immunohistochemistry shows that Helicobacter pylori can colonize the gastric mucosa at

dogs; mmunohistochemical method based on policlonal antibody anti Helicobacter pylori can make the difference between Helicobacter species; proved by non-marked with brown chromogen of Helicobacter felis;

� There is a direct correlation between gastric infection grade; histopathological lesions severity and positive reaction for Helicobacter pylori at immunohistochemistry;

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� Predominant histopathological lesions which were found consist in: chronic follicular gastritis; acute stomach ulcer; chronic gastritis with mononuclear cells infiltrate and atrophyc glands;

� Immunohistochemistry performances must be correlated with other methods which have a great specificity and sensibility; such as PCR metod and In situ Hybridization.

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