Histological and immunohistochemical detection of different Helicobacter species in the gastric mucosa of cats

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1 J Vet Diagn Invest 13:3 12 (2001) Histological and immunohistochemical detection of different Helicobacter species in the gastric mucosa of cats Eugenio Scanziani, Kenneth W. Simpson, Silvia Monestiroli, Sabina Soldati, Dalit Strauss-Ayali, Fabio Del Piero Abstract. Detailed histopathological evaluation of the gastric mucosa of Helicobacter-infected cats is complicated by the difficulty of recognizing Helicobacter organisms on hematoxylin and eosin (HE)-stained sections and the ability of multiple Helicobacter species to infect cats. In this study, the presence and localization of different species of Helicobacter in the stomachs of cats was investigated using silver staining and immunohistochemistry. Five groups containing 5 cats each were established (group 1: urease negative and Helicobacter free; groups 2, 3, 4, and 5: urease positive and infected with Helicobacter heilmannii, unclassified Helicobacter spp., Helicobacter felis, and Helicobacter pylori, respectively). Gastric samples were evaluated by HE and silver staining and by immunohistochemistry with 3 different anti-helicobacter primary antibodies. Helicobacter were detected by Steiner stain in all infected cats at the mucosal surface, in the lumen of gastric glands, and in the cytoplasm of parietal cells. In silver-stained sections, H. pylori was easily differentiated from H. felis, H. heilmannii, and unclassified Helicobacter spp., which were larger and more tightly coiled. No organisms were seen in uninfected cats. Helicobacter antigen paralleled the distribution of organisms observed in Steinerstained sections for 2 of the 3 primary antibodies tested. The antisera were not able to discriminate between the different Helicobacter species examined. A small amount of Helicobacter antigen was present in the lamina propria of 3 H. pylori-, 3 H. felis-, and 1 H. heilmannii-infected cat. Minimal mononuclear inflammation was present in uninfected cats and in those infected with unclassified Helicobacter spp. and H. heilmannii cats. In H. felis-infected cats, lymphoid follicular hyperplasia with mild pangastric mononuclear inflammation and eosinophilic infiltrates were present. The H. pylori-infected cats had severe lymphoid follicular hyperplasia and mild to moderate mononuclear inflammation accompanied by the presence of neutrophils and eosinophils. These findings indicate that Steiner staining and immunohistochemistry are useful for detecting Helicobacter infections, particularly when different Helicobacter species can be present. Monoclonal antibodies specific for the different Helicobacter species could be important diagnostic aids. There appear to be differences in the severity of gastritis in cats infected with different Helicobacter species. Gastric spiral-shaped bacteria of the genus Helicobacter have been described in many animal species as well as in humans. 24,81 Although their presence has been documented since the end of the 19th century, it is only since the discovery of Helicobacter pylori in humans by Warren and Marshall in 1983 that much interest has been expressed in gastric Helicobacter in animals. 5,74,77,87 Helicobacter pylori infection in humans has been associated with chronic active gastritis, peptic ulcer, gastric mucosa associated lymphoid tissue lymphoma, and adenocarcinoma. 10,20,30,31,41,69,70,90 An association between the presence of Helicobacter infection and gastritis has been described in several From the Università degli Studi di Milano, Facoltà di Medicina Veterinaria, Istituto di Anatomia Patologica Veterinaria e Patologia Aviare, Milano, Italy (Scanziani, Soldati), The College of Veterinary Medicine, Cornell University, Ithaca, New York (Simpson, Strauss-Ayali), The Istituto Nazionale dei Tumori, Milano, Italy (Monestiroli), and the Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, Kennett Square, Pennsylvania (Del Piero). Received for publication December 13, animal species. 6,7,13 15,21,46,47,50,51,54,59,65,67,73,75 The potential role of Helicobacter in the development of gastric adenocarcinoma has been reported in naturally infected ferrets and experimentally infected Mongolian gerbils. 22,88 A possible zoonotic role for some Helicobacter species has also been suggested. 32,52,67,84,89 Infection with Helicobacter spp. is common in domestic cats with reported prevalence rates ranging between 41% and 100%. 28,32,33,39,64,67 Helicobacter felis and Helicobacter heilmannii-like organisms are the most common species infecting cats. 44,64,66 Helicobacter felis has been definitively classified, but a complete characterization for H. heilmannii is still lacking because it has not been isolated on artificial media. 53,71,82 Thus, H. heilmannii is a provisional name given to closely related, if not identical, organisms characterized by the absence of periplasmatic fibrils. Other than the cat, H. heilmannii-like organisms have been detected in dogs, nonhuman primates, cheetahs, swine, rats, and humans. 3,8,15,29,37,58,67,72,75 Recently, 2 H. heilmannii-related organisms have been cultured from dog stomachs and named Helicobacter bizzozeronii and 3

2 4 Scanziani et al. Figure 1. Helicobacter pylori (left) and H. felis (right) organisms in the lumina of gastric glands of cats. The differences in sizes and shapes between the 2 Helicobacter species are evident. Steiner stain, 1,120X. Helicobacter salomonis. 34,43 Other gastric Helicobacter organisms detected in cats include H. pylori and Helicobacter pametensis. 32,33,64 The detailed evaluation of gastric mucosal lesions and the detection of Helicobacter organisms in histological sections is an important contribution to understanding the pathogenesis of the infection. However, Helicobacter are not easily recognized on routine hematoxylin and eosin (HE)-stained sections. 2,4,75 Specific silver staining with Warthin Starry, Genta, or Steiner methods are widely employed to detect Helicobacter. 9,19,27,32,33,35,58,64,78 The presence of multiple Helicobacter species that can colonize the gastric mucosa of the cat complicates the investigations of the role of Helicobacter in the pathogenesis of gastric diseases in cats. Two main groups of Helicobacter can be distinguished morphologically on silver-stained histological sections. The first group includes species similar to H. pylori: 2 4 m long, with few, loose helical turns. The second group includes larger spiral-shaped Helicobacter species, such as H. felis and H. heilmannii-like organisms, which are 7 10 m long, with a tightly wound spiral shape. 42,75 Immunohistochemistry for H. pylori is considered a useful diagnostic tool in human pathology; it is highly specific and has a higher sensitivity than bacteriological culture and other classical histological stainings methods. 2,4,45,55 Immunohistochemistry is sensitive enough to identify low numbers or even single organisms and is more accurate than other diagnostic meth- Figure 2. Large Helicobacter organisms fill the lumen of a gland in the pyloric mucosa of a cat infected with an unclassified Helicobacter species. Steiner stain, 700X.

3 Gastric Helicobacter in cats 5 Figure 3. Helicobacter organisms are detectable within the cytoplasm of a parietal cell in the fundic mucosa of a cat infected with H. pylori. Steiner stain, 1,000X. ods in identifying H. pylori after specific therapy. 57 Immunohistochemistry has been infrequently employed as a diagnostic assay or aid in studies of the pathogenesis of gastric Helicobacter infection in veterinary medicine. 42,58,75 In the present study, we investigated the density of colonization, localization, and histopathological consequences of infection with H. heilmannii, large unclassified Helicobacter spp., H. felis, and H. pylori in the gastric mucosa of cats using silver and HE staining and immunohistochemistry. Materials and methods Animals. Twenty-five cats were studied. The presence or absence of gastric Helicobacter spp. was ascertained in all cats by evaluating gastric biopsies for urease activity and the presence of Helicobacter spp. DNA by polymerase chain reaction (PCR). All infected cats had positive biopsy urease tests and were positive with Helicobacter genus-specific primers. Uninfected cats were urease and PCR negative. The cats were assigned to the following 5 groups on the basis of genus- and species-specific PCR for Helicobacter. Group 1 included 5 specific-pathogen-free, Helicobacter-free cats (1.6 yr old, males) obtained from a commercial vendor. a Group 2 included 5 cats (3 females, 2 males) with naturally acquired H. heilmannii infection. Four cats were strays of undetermined age presented for spay/neuter. One cat was a 12-yr-old castrated male that underwent endoscopy for investigation of weight loss, diarrhea, and vomiting. Group 3 included 5 cats (4 females, 1 male) naturally infected with unclassified Helicobacter spp. These cats were colonized by Figure 4. Black, punctate debris particles of various sizes are detectable within the cytoplasm of parietal cells in the fundic mucosa of a cat infected with H. felis. Steiner stain, 850X.

4 6 Scanziani et al. large gastric spiral organisms that were positive with genusspecific Helicobacter PCR but negative for H. pylori, H. heilmannii, and H. felis species-specific PCR. Four cats were stray cats of undetermined age presented for spay/neuter. One cat was a 12-yr-old female that had endoscopy for investigation of diarrhea and suspected liver disease. Group 4 included 5 cats (1.6 yr old, males) 1 yr after oral inoculation with H. felis ATCC Group 5 included 5 cats (0.5 yr old, males) from a colony known to be infected with H. pylori. Cats from groups 1, 4, and 5 were experimental cats undergoing studies to evaluate the effect of Helicobacter colonization on gastric function. They were housed in a barrier facility, BL2 (Baker Institute for Animal Health, Cornell University, Ithaca, NY), with a controlled environment (lighting 12 hr on, 12 hr off; temperature C, humidity 35 45%). Each group of cats was housed separately. Cats from groups 1, 4, and 5 were euthanized at the end of the experiment with an intravenous overdose of pentobartibal sodium. Cats from groups 2 and 3 were either stray cats admitted for spay/neuter (8 cats) or cats undergoing diagnostic endoscopy (2 cats). Cornell University operates under an approved Animal Welfare Assurance (A ) and is fully accredited by the American Association for the Accreditation for Laboratory Animal Care. The project was approved by the Institutional Animal Care and Use Committee at Cornell University. Gastric tissue sampling. Endoscopic biopsies of the gastric mucosa were obtained from cats in groups 2 and 3 with a pediatric endoscope b and biopsy forceps. Cats were sedated with ketamine (10 mg/kg intramuscularly), intubated, and maintained on isofluorane and oxygen. Endoscopic biopsies were procured from the cardia, the stomach body (greater curvature), and the pyloric antrum (incisura to pyloric sphincter). Three biopsies were taken from each site for histology, 2 from each site for urease testing, and 1 from each site for PCR. Endoscopic biopsy samples for PCR analysis were placed in sterile tubes and kept on ice until frozen at 80 C pending analysis. The endoscope was thoroughly cleaned and then sterilized using an activated aldehyde solution. c Biopsy forceps were sterilized in a similar fashion, and the biopsy cups were immersed in sodium hypochloride (1:10 in water) for 10 min to destroy residual DNA. Full-thickness gastric samples were obtained from cats in groups 1, 4, and 5 at necropsy from 10 standardized sites as previously described, using a 6-mm skin biopsy punch. 54 One sample each from the cardia (site 1), midbody (site 5), and midpylorus (site 8) was evaluated for urease activity and frozen for PCR analysis. Pooled tissue samples from the cardia, body, and fundus (sites 1 6) and the pyloric antrum (sites 7 10) were evaluated histologically. Gastric urease. Urease production by gastric tissue was evaluated as previously described. 73 Gastric tissue samples were placed in sterile tubes containing 200 l of a solution composed of urea, sodium azide, phenol red, and phosphatebuffered saline (ph 6.5). A change from orange-red to bright pink was considered a positive result. PCR. DNA was extracted from gastric samples using a commercially available kit. d PCR was performed using Helicobacter genus-specific primers C97 and C05 to test for 16S ribosomal RNA (rrna) amplicons 80 and using primers that amplify the urease B genes of H. felis, H. heilmannii, and H. pylori 23,64 as previously described. Validation employed H. pylori (Cornell cat strain 1, ATCC 49503, ATCC 43504, human isolate 8826), H. felis (ATCC 49179), H. bizzozeronii (ATCC ), H. salomonis (CCUG 37845), H. heilmannii (DNA from the stomach of an infected cat), H. fenelliae (ATCC 35684), H. bilis (ATCC 51630), H. cinaedii (ATCC 35683), H. hepaticus (ATCC 51450), and H. canis (ATCC 51401). No amplicons were obtained with DNA from Campylobacter jejuni (dog isolate) and Proteus mirabilis (cat isolate). Annealing temperatures for H. felis, H. pylori, and H. heilmannii were 55 C, 57 C, and 57 C, respectively. Single bands at 1,150 bp, 1,707 bp, and 580 bp were present when H. felis, H. pylori, and H. heilmannii were used, respectively. Histopathology. Tissue samples were fixed in 10% buffered formalin, routinely processed for paraffin embedment, sectioned at 5 m, and stained with HE. A modified Steiner stain was used on replicate sections for the detection of Helicobacter organisms. 25 Pooled tissue samples from the cardia, body, fundus (fundic mucosa), and pyloric antrum (pyloric mucosa) were evaluated by light microscopy. The histological sections were entirely examined. The exam was done in a blind fashion. The number of Helicobacter organisms in Steiner-stained sections was scored as follows: absence of Helicobacter organisms; presence of Helicobacter organisms in 5% of the gastric glands; presence of Helicobacter organisms in 5 50% of the gastric glands; presence of Helicobacter organisms in 50% of the gastric glands. All glands in the entire section were examined for Helicobacter organisms. Immunohistochemistry. Histological sections were stained by the avidin biotin peroxidase complex procedure with a commercial immunoperoxidase kit. e,40 Sections were dewaxed, treated with 0.5% hydrogen peroxide in methanol for 20 min, and rehydrated. Sections were incubated with the primary antibody at 4 C for 18 hr. Three different commercially available primary antibodies were employed: 1) polyclonal rabbit antibody to H. pylori (PAb Hp) f at a working dilution of 1:1,000; 2) mouse monoclonal antibody (MAb) to Campylobacter jejuni cross-reacting with H. pylori, clone 371/ (MAb 371/254.55) g at a working dilution of 1:1,000; and 3) mouse MAb to H. pylori, clone 6611 (MAb 6611) h at a working dilution of 1:500. After washing, the sections were covered with a 1:200 dilution of goat anti-rabbit immunoglobulin-biotinylated secondary antibody and left at room temperature for 30 min. The peroxidase-conjugate ABC was allowed to react at room temperature for 30 min. Sections were incubated with 0.05% diaminobenzidine and 0.01% hydrogen peroxide substrate for 3 min, washed in tap water, and counterstained with Mayer hematoxylin. Negative controls for each sample were prepared by replacing the primary antibody with normal rabbit or mouse serum. Known negative and positive control sections were included in each immunolabeling assay. Results Silver staining. Helicobacter organisms were detected by the Steiner stain in all Helicobacter-infected

5 Gastric Helicobacter in cats 7 Figure 5. Degree of Helicobacter colonization in fundic and pyloric mucosa in 4 groups of infected cats. The presence and number of organisms has been evaluated histologically in Steiner stained sections and scored as follows: absence of organisms; presence of organisms in 5% of the gastric glands; presence of organisms in 5 50% of the gastric glands; presence of organisms in 50% of the gastric glands. Each line represents a single cat infected with H. heilmannii (A), unclassified Helicobacter spp. (B), H. felis (C), or H. pylori (D). cats (groups 2 5), but no organisms were seen in uninfected cats (group 1). In H. pylori-infected cats (group 5), the organisms were comma shaped, loosely coiled, and 2 3 m long. They were easily differentiated from the organisms detected in cats infected with H. heilmannii (group 2), unclassified Helicobacter spp. (group 3), and H. felis (group 4); the organisms were larger, more tightly coiled, and up to 8 m long (Fig. 1). The morphological appearance of the large spiral bacteria observed in cats from groups 2 4 was similar. In all infected cats, Helicobacter organisms could be detected at the mucosal surface and in the lumina of gastric glands (Fig. 2). Helicobacter organisms were also detected in the cytoplasm of parietal cells of the fundic mucosa (Fig. 3). Another common finding in Steiner-stained sections from infected cats was the presence of black punctate debris ranging in size from barely visible to 2 m in the cytoplasm of parietal cells (Fig. 4). The degree of colonization in the different groups of infected cats is shown in Fig. 5. In all infected groups the number of organisms was high in the fundic mucosa, particularly in the cats infected with unclassified Helicobacter spp. The number of organisms was variable in the pyloric mucosa. In general, higher numbers of organisms were present in fundic mucosa than in the pyloric mucosa in all cats except those infected with H. pylori, in which a similar number of organisms was found for these 2 sites (Fig. 5). In 11 cats, fewer organisms were detected in the pyloric than in the fundic mucosa; in 2 of these cats, Helicobacter organisms were not detected in the pyloric mucosa. In 7 cats, there were similar numbers of organisms in the pyloric and fundic mucosa. In only 2 cats was the number of organisms higher in the pyloric than in the fundic mucosa. Immunohistochemistry. Immunohistochemical staining with PAb Hp and MAb 371/ provided similar results and was positive in all Helicobacterinfected cats. Control uninfected cats were negative. Positive staining was characterized by a coarse granular brown precipitate that was easily detectable against the light blue background. The morphology of single organisms with immunohistochemistry was not as well appreciated as in Steiner-stained sections. The localization of Helicobacter antigen with immunohistochemistry paralleled the distribution of organisms in Steiner-stained sections with positive immunolabeling at the mucosal surface (Fig. 6), in the lumina of glands, and in the cytoplasm of parietal cells in the fundic mucosa. In the cytoplasm of parietal cells, positivity was characterized by the presence of punctate debris ranging in size from barely visible to 2 m that stained positively with the primary antibodies (Fig. 7). Moreover, in 3 H. pylori-, 3 H. felis-, and 1 H. heilmannii-infected cat, a small amount of Helicobacter antigen was present in the lamina propria within areas of severe leukocyte infiltration or within lymphoid follicles (Fig. 8). Positive immunolabeling was detected with the MAb 6611 in the lumina of gastric glands in 2 of the cats infected with the unclassified Helicobacter spp. A weak and inconsistent positive staining of cytoplasmatic punctate debris was noted in 4 of 5 H. pyloriinfected cats and in the 2 cats infected with the unclassified Helicobacter spp. that showed luminal positivity. A weak background staining was present in superficial epithelial cells and parietal cells in all cats infected with H. pylori, H. heilmannii, and H. felis, in 4 out of 5 cats infected with the unclassified Helicobacter spp., and in 4 of 5 control uninfected cats. Histopathology. Microscopic lesions in uninfected cats (group 1) were minimal. They consisted of rare

6 8 Scanziani et al. Figure 6. Clusters of immunolabeled organisms at the surface of pyloric mucosa of a cat infected with an unclassified Helicobacter spp. Immunohistochemical staining for Helicobacter (primary antibody: MAb 371/254.55), hematoxylin counterstain, 400X. and minimal inflammatory infiltrates composed of lymphocytes and plasma cells in 4 cats, 1 of which had rare lymphoid follicles in pyloric mucosa. Minimal fibrosis and, in 2 cats, atrophy of pyloric mucosa were also present. Similar microscopic lesions were present in 4 of 5 H. heilmannii-infected cats and in cats infected with unclassified Helicobacter spp. In the remaining H. heilmannii-infected cat, moderate lymphoid follicular hyperplasia in pyloric mucosa and moderate inflammatory infiltrates composed of lymphocytes, plasma cells, and macrophages were present in fundic and pyloric mucosa. This cat was 12 years old and had chronic vomiting, diarrhea, and weight loss. Lesions observed in H. felis-infected cats were characterized by lymphoid follicular hyperplasia in all cats (mild in 1 cat, moderate in 2 cats, and severe in 2 cats) and mild but diffuse (pangastric) inflammatory infiltrates composed of lymphocytes, plasma cells, macrophages, and eosinophils in 3 cats. Mild to moderate, diffuse fibrosis and atrophy of the pyloric mucosa were also present in all cats. All H. pylori-infected cats had severe lymphoid follicular hyperplasia in the pyloric mucosa. Inflammatory infiltrates (mild in 2 cats and moderate in 3 cats) consisting of lymphocytes, plasma cells, macrophages, eosinophils, and neutrophils were observed, with the pyloric mucosa more severely affected than the fundic mucosa. Mild to moderate, diffuse fibrosis and, in 3 cats, atrophy of pyloric mucosa were also present. Figure 7. Intracytoplasmatic Helicobacter-positive antigen in a parietal cell of fundic mucosa of a cat infected with H. felis. Immunohistochemical staining for Helicobacter (primary antibody: MAb 371/254.55), hematoxylin counterstain, 1,120X.

7 Gastric Helicobacter in cats 9 Figure 8. Helicobacter antigen is present within an area of inflammation in the pyloric mucosa of a cat infected with H. felis. Immunohistochemical staining for Helicobacter (primary antibody: MAb 371/254.55), hematoxylin counterstain, 300X. Discussion Silver staining was sensitive and specific for identification of Helicobacter-infected cats. Silver stains are capricious and frequently have artifacts that can be mistaken for bacteria. 85 However, with most Helicobacter infections the number of organisms is usually very high, and the morphology of the bacteria is so characteristic that they can be easily and specifically detected. 25,49,58,75 Steiner stain clearly differentiated H. pylori from H. felis, H. heilmannii, and unclassified Helicobacter spp. The ability to distinguish H. pylorilike organisms from larger spiral-shaped Helicobacter organisms with silver staining has been reported in cynomolgus monkeys with concurrent H. pylori and H. heilmannii infections. 75 Intracytoplasmatic organisms were clearly detected in parietal cells from all Helicobacter-infected cats. The presence of large spiralshaped Helicobacter organisms within the cytoplasm of parietal cells is a common finding in several animal species, such as rhesus monkeys, cynomolgus monkeys, rats, cats, and dogs. 12,29,35,38,39,75 Electron microscopic observations indicate that these organisms are within dilated intracellular canaliculi of parietal cells. 28 Unlike large Helicobacter organisms in these animal species, H. pylori in humans is not considered an invasive pathogen and has been rarely described within parietal cells. 76,83 The black intracytoplasmatic debris observed with silver staining in parietal cells was also positive for Helicobacter antigen. The various sizes of these particles suggest that they represent degraded Helicobacter organisms. An intracellular localization of H. pylori could protect organisms from antibacterial treatments. 17,18 Colonization of fundic mucosa by H. felis, H. heilmannii, and unclassified Helicobacter spp. was dense and was more extensive than that in the pyloric mucosa. A similar finding was observed in cats and dogs after natural infection with Helicobacter spp. and experimental infection with H. felis. 36,79,80,91 The reasons for this difference in colonization density are unclear. Differences in the microenvironmental conditions from the different sites of the gastric mucosa may be a factor. 86 Another possible explanation is that bacterial colonization is enhanced in gastric areas with less prominent mucosal cellular inflammatory response. 26,27 This hypothesis is supported by the observation that lymphoid follicular hyperplasia was evident in the pyloric mucosa of H. felis-infected cats despite the fact that this site was less densely colonized. In contrast, H. pylori densely colonized both the pyloric and fundic mucosa, despite marked cellular inflammatory response in the pyloric mucosa. Immunohistochemistry for Helicobacter antigen with 2 of the primary antibodies tested in this study (PAb Hp and MAb 371/254.55) was highly sensitive and produced positive results in all infected cats. Immunohistochemistry was not able to discriminate among H. pylori, H. felis, H. heilmannii, and unclassified Helicobacter spp.; all these species were stained. These 2 primary antibodies are also able to immunostain other Helicobacter species, including H. hepaticus and H. heilmannii-like organisms from the stomach of cynomolgus monkeys, dogs, pigs, and rats (unpublished observations). Other authors have reported the immunohistochemical staining with PAb Hp and MAb 371/ or other anti-h. pylori antibodies of H. heilmannii-like organisms and H. pylori from gastric mucosa of cynomolgus monkeys. 58,75 Bacteria morphologically similar to H. heilmannii and H. felis stained positively with a rabbit PAb directed against

8 10 Scanziani et al. H. pylori. 42,75 These observations indicate a high degree of antigenic homology among the different Helicobacter species. A broad spectrum of immunohistochemical reactivity may be considered useful in the generic detection of Helicobacter infections, particularly for diagnostic use in animal species such as the cat, in which different Helicobacter species can be present. Helicobacter species-specific MAbs could provide important diagnostic aids. No satisfactory results were obtained with the MAb 6611 under the conditions employed in this study. A nonspecific background staining was present in most of the infected and noninfected cats. It is possible that gastric epithelial cells bound the primary antibody because of some antigenic homology between bacterial and host antigens. 63 In this study, Helicobacter antigen was detected in the lamina propria of cats infected with H. pylori, H. felis, and H. heilmannii. A similar finding has been reported in humans with an active H. pylori infection. 1 More recently, large spiral-shaped Helicobacter organisms were histologically detected by the Genta stain beneath the surface gastric epithelium and within the lymphoid follicles in cats. 78 The presence of Helicobacter antigen in the lamina propria may be an important factor in the induction and development of a gastric inflammation. Histological findings in the stomachs of Helicobacter-infected animals range from normal to severe inflammation, apparently dependant on the species and strain of Helicobacter involved, the species and strain of the host, and the duration of the infection. 62,75 In the current study, H. pylori-infected cats showed signs of chronic gastric inflammation characterized by severe lymphoid follicular hyperplasia and minimal to moderate mononuclear inflammation, accompanied by the presence of granulocytes. These findings are broadly similar to those for H. pylori-infected humans, particularly children in whom gastritis is characterized by mononuclear cells and lymphoid follicles, with less severe polymorphonuclear cell infiltrates than adults. 11,16,30,56,60,61 In H. felis infected cats, the lesions were characterized by lymphoid folliclular hyperplasia and mild but pangastric mononuclear inflammation with eosinophils. 80 Mononuclear inflammation of minimal intensity was present in cats infected with unclassified Helicobacter spp. and in most of the H. heilmannii-infected cats. The spectrum of changes observed in cats colonized with large gastric Helicobacter spp. in the present study is similar to that reported in cats, captive exotic carnivores, and small felids naturally infected with Helicobacter. 32,36,39,48 Although the differences in histopathological findings may be related to the species of Helicobacter, factors such as the size of tissue sample examined, the age of the cat, and the presence or absence of clinical signs also must be considered. Differences in the size of the tissue samples taken from H. pylori- and H. felis-infected cats (6 mm) versus those taken from cats infected with H. heilmannii and unclassified Helicobacter spp. (about 2 mm) may have lead to the underestimation of the extent and severity of lesions in these 2 groups of infected cats. 64 Researchers who have evaluated fullthickness gastric tissue samples rather than endoscopic biopsies have found a relationship of Helicobacter infection to lymphoid follicular hyperplasia in cats. 30,39,64,66 68,78,91 The H. heilmannii-infected cat with gastritis was a 12-year-old castrated male undergoing endoscopy for investigation of weight loss, diarrhea, and vomiting. Minimal signs of gastric inflammation similar to those observed in cats infected with unclassified Helicobacter spp. or H. heilmannii were also present in uninfected control cats. This finding indicates that factors other than Helicobacter may induce a gastric inflammatory reaction. 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