Localization of Helicobacter spp. in the fundic mucosa of laboratory Beagle dogs: an ultrastructural study

ABSTRACT: In dogs Helicobacter spp. are found in all gastric regions usually localized in the surface mucus, gastric glands and parietal cells. The aim of this study was to detail the distribution of Helicobacter spp. in the fundic mucosa of asymptomatic Beagle dogs and their intracellular localization within parietal cells, in order to evaluate species-specific pathogenetic effects on gastric cells. The presence of Helicobacter spp. was investigated by immunohistochemistry, TEM, and PCR in the fundic mucosa of six Beagle dogs. Helicobacter spp. were found in all dogs examined, and H. bizzozeronii and H. felis were identified by PCR and confirmed by TEM. In the lumen of the fundic glands, co-localization was common. H. bizzozeronii was present in larger numbers than H. felis in both intraluminal and intraparietal localization. The amounts of H. bizzozeronii were similar in superficial and basal portions of the glands. H. felis was predominantly localized in the superficial portions of gastric glands but almost absent from the base. Within parietal cells, most Helicobacter organisms were intracanalicular, but intact and degenerate Helicobacter organisms were also visualized free in the cytoplasm or in secondary lysosomes. No specific degenerative lesions were found in infected parietal cells. Helicobacter organisms were also observed within macrophages in the lamina propria. In conclusion, there is a differential distribution of H. bizzozeronii and H. felis in the fundic mucosa of Beagle dogs, and their intracellular localization in parietal cells and macrophages suggests novel pathogenic scenarios for the development of immune response and maintenance of chronic gastritis in dogs.     

A spiral microorganism in the stomach of pigs.

Light and electron microscopic and microbiologic evaluations were performed on mucosa of stomachs from 120 healthy slaughtered pigs. Helicobacter pylori was not found, but a tightly spiralled bacterium, not previously described, was seen in histological sections and/or in carbol fuchsin stained smears in 13 (10.8%) stomachs. In paraffin sections stained with carbol fuchsin, the bacteria were seen in the mucus of the lumen of the antral pits and in the mucosa surface within and beneath the mucus. In this sections of Polilyte embedded tissue the bacteria had three to eight spiral turns per cell (mean = five), flattened ends, a Gram-negative cell-wall structure and a sheathed flagella. The urease test was positive in gastric mucosa of 13 bacteria-positive pigs (10.8%). The microorganism was not cultured and did not cross-react with polyclonal antibodies raised in rabbits against H. pylori. Superficial chronic gastritis and "borderline" gastritis were observed in antral mucosa of 10 (76.9%) and of two (15.4%) spiral bacteria-positive pigs, respectively.     

Histological studies of the occurrence of organisms shaped like Campylobacter in the abomasum of calves]

The mucosa of the abomasum is a strongly acidic and therefore germ poor, but not entirely germfree environment. Only in isolated areas of the fundic glands zone were straight rods in the foveolas, and even more seldom spiral shaped bacteria could be encountered. They had no relation to the inflammatory infiltrates. On the other hand, Campylobacter-like organisms were more often observed in the pyloric region and, preferably, in a narrow zone following the cutaneous mucosa of the omasum. These organisms appeared to have a different size (1.3 to 2.4 microns length and 0.4 to 0.8 microns width) and they occurred deep inside the glands in an almost pure fashion. Whereas they caused no visible reaction of the tissue in the narrow cardiac zone, their occurrence in the pyloric region was several times connected with neutrophilic-rich infiltration. It ought to be tested, whether Helicobacter are among these Campylobacter-like organisms, which cause a disease leading up to ulcerations in calves similar to gastritis B in humans.     

Role of Helicobacter felis in chronic canine gastritis.

Five gnotobiotic Beagle dogs were orally inoculated with a pure culture of Helicobacter felis. The remaining two littermates served as contact controls. Thirty days after infection, all animals were euthanatized and specimens were collected for evaluation. In infected dogs, H. felis was recovered from all areas of the stomach. Colonization was heaviest in the fundus and antrum. H. felis was not cultured from any segment of the gastrointestinal tract distal to the duodenum. Two weeks after infection, all five infected dogs had detectable IgM and IgG serum antibody to H. felis, whereas control dogs had no measurable H. felis serum antibody throughout the study. Histopathologic changes in the stomachs of infected dogs included large numbers of lymphoid nodules throughout all regions of the gastric mucosa and were most numerous in the fundus and body. A mild, diffuse lymphocytic infiltrate with small numbers of plasma cells and eosinophils was also present in the subglandular region of all portions of the gastric mucosa. Electron microscopic examination revealed large numbers of spiral-shaped H. felis in gastric mucus adjacent to or superimposed over the areas of inflammation. Occasionally, however, H. felis was observed within the canaliculi of gastric parietal cells. Histopathologic changes in the stomachs of the contact control dogs were limited to focal infiltrates of eosinophils and small aggregates of lymphocytes in the subglandular portions of the gastric mucosa in one animal. Infection with H. felis is a likely cause of naturally occurring lymphofollicular gastritis.     

Morphological,morphometric and histochemical characterization of the gastric mucosa of the camel (Camelus dromedarius)

Mucosal morphology, morphometry and mucin histochemistry of the stomach were studied in the one-humped camel. The lining of the stomach was divided into eight grossly identifiable regions. The first region was non-glandular, occupied the body of the first compartment of the stomach and constituted 53.2% of the gastric mucosa. The other seven regions were lined by a glandular mucosa. Histological, histochemical and morphometric investigations have shown that glandular mucosa comprises pseudo-cardiac, cardiac, fundic and pyloric regions. The pseudo-cardiac region was characterized by widely separated short tubular serous glands. It constituted 36.2% of the gastric mucosa; it extended over the entire lining of the second compartment and parts of the first and third compartments. The cardiac region was confined to the initial zone of the third compartment amidst the psuedo-cardiac region but contiguous with the distal end of the gastric groove. It constituted 3.4% of the gastric mucosa and was characterized by neutral and acid mucin positive glands. The fundic and pyloric regions occupied the distal distended part of the third compartment. The fundic region constituted 4.3% of the gastric mucosa. It was packed with typical fundic glands characterized by chief cells, parietal cells and acid mucin positive neck cells. The pyloric region constituted 2.9% of the gastric mucosa. Its glands were positive to acid mucins except for their bases that were positive to neutral mucins. Differences in volume densities of the mucosal components and reactivity of the surface epithelium and gastric pits to mucin stains were noted in the different regions of the stomach.     

Genome sequence of Helicobacter suis supports its role in gastric pathology

ABSTRACT: Helicobacter (H.) suis has been associated with chronic gastritis and ulcers of the pars oesophagea in pigs, and with gastritis, peptic ulcer disease and gastric mucosa-associated lymphoid tissue lymphoma in humans. In order to obtain better insight into the genes involved in pathogenicity and in the specific adaptation to the gastric environment of H. suis, a genome analysis was performed of two H. suis strains isolated from the gastric mucosa of swine. Homologs of the vast majority of genes shown to be important for gastric colonization of the human pathogen H. pylori were detected in the H. suis genome. H. suis encodes several putative outer membrane proteins, of which two similar to the H. pylori adhesins HpaA and HorB. H. suis harbours an almost complete comB type IV secretion system and members of the type IV secretion system 3, but lacks most of the genes present in the cag pathogenicity island of H. pylori. Homologs of genes encoding the H. pylori neutrophil-activating protein and γ-glutamyl transpeptidase were identified in H. suis. H. suis also possesses several other presumptive virulence-associated genes, including homologs for mviN, the H. pylori flavodoxin gene, and a homolog of the H. pylori vacuolating cytotoxin A gene. It was concluded that although genes coding for some important virulence factors in H. pylori, such as the cytotoxin-associated protein (CagA), are not detected in the H. suis genome, homologs of other genes associated with colonization and virulence of H. pylori and other bacteria are present.     

Association of tightly spiraled bacterial infection and gastritis in pigs

Tightly spiral bacteria were observed only in the pyloric mucosa of 4 (8.0%) of 50 swine stomachs, mainly in the surface of epithelia, the gastric pits and the lumen of gastric glands. The presence of the spiral bacteria was significantly associated with chronic pyloric gastritis (p<0.05). Mean gastritis score of the bacteria-positive pyloric mucosa was 3.25 +/- 0.25, whereas that of the bacteria-negative pyloric mucosa was 2.37 +/- 0.12. Parakeratosis and hyperkeratosis were spontaneously seen in the mucosa layer of pars oesophagea, regardless of the bacterial infection. Marked infiltration of mononuclear cells and granulocytes were seen in the cardiac mucosa, regardless of the bacterial infection. Mean gastritis score of the bacteria-positive cardiac mucosa was 3.27 +/- 0.32, whereas that of the bacteria-negative cardiac mucosa was 2.84 +/- 0.13. There was no significant difference between the bacteria-positive and negative cardiac mucosa (p>0.05). Inflammatory response in the fundic mucosa was rare (gastritis score=0.75 +/- 0.08). The tightly spiraled bactera were not cultured with various culture media. These results suggest that the presence of tightly spiraled bacteria is associated with only the pyloric gastritis in pigs.     

The diagnosis of gastritis and helicobacter-like organisms infection in endoscopic biopsies of the canine gastric mucosa

The aim of this study was to evaluate the prevalence of gastric Helicobacter-like organisms (GHLO) and gastritis in the gastric mucosa of dogs with gastric disorders. Tissue samples of the gastric mucosa were obtained from 30 dogs with gastrointestinal symptoms (vomiting, abdominal pain or discomfort, loss of appetite) during endoscopy. Histopathological examinations were performed and occurrence of GHLO infection, gastritis and other mucosal changes were estimated. The GHLO infection and gastritis were identified in 63.3 and 36.6% of dogs respectively; other mucosal changes included fibrosis in the lamina propria, degenerative changes of the gastric glands and hyperplasia of the parietal cells. The present study has revealed that microscopically found gastritis is not frequent in dogs examined by endoscopy. GHLO infection can be responsible for some cases of gastritis and hyperplasia of parietal cells in dogs.     

The high prevalence of Helicobacter sp. in porcine pyloric mucosa and its histopathological and molecular characteristics

This study examined the prevalence of Helicobacter infection in the pyloric mucosa of pigs and its histopathological and molecular characteristics. Forty porcine pyloric samples were examined for Helicobacter infection by silver staining and PCR assay. The PCR product (376 bp) was digested with NdeII to differentiate between Helicobacter heilmannii and Helicobacter pylori. Another PCR assay run to produce an 1157 bp fragment was performed using a primer set designed from the 16S rRNA gene of Candidatus H. suis, and its product was cloned and sequenced. Infection rates were 62.5% (25/40) and 95.0% (38/40) as determined by silver staining and the PCR assay, respectively. On histopathological examination, lymphoid follicle aggregation in the pyloric mucosa and granulocytic migration into the lumen of pyloric glands were observed in 24 (60.0%) and 33 (82.5%) gastric samples, respectively. All PCR products, except that of H. pylori, were cut into two fragments of 147 and 229 bp by enzymatic digestion with NdeII. Sequencing of the 16S rRNA gene showed that the bacterium had 99.57% (1152 bp/1157 bp) homology to the 16S rRNA gene of Candidatus H. suis.     

Experimental Infection of Pigs with ‘<Emphasis Type="Italic">Candidatus</Emphasis> Helicobacter suis’

‘Candidatus Helicobacter suis’ is a spiral-shaped bacterium that colonizes the stomach of more than 60% of slaughter pigs. The role of ‘Candidatus Helicobacter suis’ in gastric disease of pigs is still unclear. Experimental studies in pigs are lacking because this bacterium is unculturable until now. An inoculation protocol using ‘Candidatus Helicobacter suis’ infected mouse stomach homogenate was used to reproduce the infection in pigs. Control animals were inoculated using negative mouse stomach homogenate. Pigs were inoculated three times with one-week intervals and euthanized 6 weeks post inoculation. Tissue samples were taken from different mucosal stomach regions to detect ‘Candidatus Helicobacter suis’ by PCR and urease test. Mucosal inflammation was evaluated on formalin-fixed tissue samples. Lesions in the pars oesophagea were scored macroscopically. Infection was succesful in all challenged animals, with the antrum and the fundus being predominantly positive. Infection was associated with infiltration of lymphocytes and plasma cells in the antral mucosa, evolving to follicular gastritis. No apparent inflammation of the fundic stomach region was detected in the infected animals. A clear link between ‘Candidatus Helicobacter suis’ and pars oesophageal lesions could not be found.     

Isolation and characterization of Campylobacter,Helicobacter, and Anaerobiospirillum strains from a puppy with bloody diarrhea

We carried out a microscopic examination of stools from a 2-month-old female puppy with bloody diarrhea, and this revealed large numbers of different spiral-shaped bacteria. To isolate these organisms, a rectal swab specimen was inoculated onto plates of Skirrow's agar and incubated at 37 degrees C for 6 days in a microaerobic atmosphere. Finally, a total of six different spiral-shaped bacteria (strains G1104, 94105, FR106, B0101, 3J102, and J2103) were isolated. Based on their morphology, biochemical traits, whole-cell protein profiles, and analysis of their 16S rDNA sequences, they were identified as Campylobacter upsaliensis, Helicobacter cinaedi, 'Flexispira rappini', two Anaerobiospirillum spp. with different morphologies, and Helicobacter sp., respectively. Analysis of 16S rRNA gene sequence data for strains 94150 (H. cinaedi) and FR106 (F. rappini) revealed that this approach has limitations when identifying isolates to the species level because of a high degree of sequence homology between these species (>99%) and considerable sequence variation among different isolates within these species. The dog was treated orally with amoxicillin for 3 days, which resolved the diarrhea. However, 1 day after the last dose the bloody diarrhea recurred but regarded to six more days amoxicillin treatment. This suggests a bacterial cause for the diarrhea. The approach to identification to microaerobic spiral-shaped bacteria in diarrheic dogs can be applied further to characterize their role in diarrhea illness.     

The first description of gastric Helicobacter in free-ranging wild boar (Sus scrofa) from Poland

Specimens of gastric mucosa of 17 free-ranging wild boars (Sus scrofa) shot in the Central Poland during 2007/2008 hunting season were investigated for the presence of Helicobacter species. Histopathology, Helicobacter genus-specific 16S rRNA PCR, and DNA sequence analysis were employed. In PCR analysis the presence of Helicobacter's DNA was detected in one stomach. Obtained sequence analysis showed its relatedness to Helicobacter heilmannii type 2. In histopathology of the PCR-positive sample the presence of tightly coiled spiral bacteria was detected on the surface of the antral mucosa, in gastric pits and lumen of the upper parts of antral glands. Potential pathologic significance of the presence of Helicobacter in the stomach of free-ranging wild boars was obscured by the parasitic invasion-caused gastritis, and remains unknown.     

Gastric helicobacters in cats.

The types of helicobacter which are found in the stomachs of carnivorous pets, especially cats, have been traditionally referred to as 'gastric helicobacter-like organisms' (GHLOs). These are microaerophilic, Gram-negative, spiral bacteria with multiple terminal flagellae and are endowed with high-level urease activity which allows them to survive in an acidic environment. Certain species have one or more periplasmic fibrils. The two GHLOs most commonly found in cats are Helicobacter felis and a species related to H heilmannii which was recently cultured from dogs. All phenotypic and genotypic (16S RNA gene sequences) evidence suggests that both of these bacteria belong in the genus Helicobacter. Whether or not helicobacters can be transmitted to humans from carnivorous pets is controversial but the recent discovery of H pylori -infected cats may be evidence of an animal reservoir for this pathogen. Although the role of H pylori in inducing antral gastritis and perpetuating pyloric ulcers in humans is well established, whether or not Helicobacter spp are causally involved in any feline gastric inflammatory conditions is unknown.     

Molecular characterization of Helicobacter pylori strains isolated from cynomolgus monkeys (M. fascicularis)

We recently reported the occurrence of natural infection with H. pylori in a group of cynomolgus monkeys with chronic active gastritis and gastric erosions. The goal of the present study was to characterize and to compare strains isolated from animals originating from two different geographical areas. Gross and microscopic pathology determined at the time of necropsy was similar in all animals. H. pylori were isolated from specimens harvested in five monkeys (four from Vietnam and one from the Philippines) with gastritis. Isolates from monkeys bred in Vietnam had a similar DNA fingerprint pattern, which was distinct from that of isolates from a monkey bred in the Philippines. All strains were of the s1a vacA subtype, but all the 'Vietnamese' strains were cagA+ and all but one were iceA1 whereas the 'Philippino' strains were cagA- and iceA2. The sequences of the 16S rRNA of the Vietnamese and Philippino strains shared 98% homology and both clustered with H. pylori sequences present in the NCBI database. In conclusion, cynomolgus monkeys can be naturally colonized by H. pylori, and the strains isolated from these animals appear to vary according to the geographical origin, thus indicating probable infection prior to importation. Since some of the cynomolgus monkeys developed antral erosions during natural infection, we propose that this animal model may be used to investigate the role of H. pylori in ulcerogenesis.     

Gastric adenocarcinoma and chronic gastritis in two related Persian cats

Two 12.5-year-old castrated male Persian cats from the same household, whose dams were littermates, presented simultaneously with gastric adenocarcinoma associated with proliferative and fibrosing gastritis. Intralesional adult Ollulanus tricuspis nematodes and rare surface-associated spiral-shaped bacteria were identified in one cat. No etiologic agents were identified in tissues from the second cat; however, gastric mucosa was examined following anthelmintic treatment. Clinical signs in each cat had commenced 2 months apart and included vomiting, hematemesis, intermittent melena, and weight loss. This is the first report of gastric adenocarcinoma occurring in housemate cats or cats of common descent. Carcinogenesis may have been influenced by shared undetermined genetic and environmental factors, possibly including Ollulanus tricuspis, spiral-shaped bacteria, or other etiologies for chronic gastritis that remain unidentified.     

Pyloric stenosis caused by hypertrophic gastritis in three dogs

Hypertrophic gastritis of the pyloric antrum is described in an 11-year-old female poodle, a 14-year-old male Maltese terrier and a 13-year-old male mongrel. The dogs suffered from chronic vomiting. Gastroscopic examination revealed mucosal proliferations in dogs 1 and 3. Radiographic examination showed signs of pyloric obstruction in all three dogs. Contrast studies demonstrated thickenings in the region of the pylorus in dogs 1 and 2. Laparotomy was done in all three dogs: in dog 1 a gastro-duodenostomy was performed and in dogs 2 and 3 the circularly thickened mucosa was resected. The mucosa of all three dogs showed hypertrophic gastritis, chiefly due to foveolar hyperplasia, and round cell infiltration, especially in the superficial layers. Herniation of mucosal glands through the muscularis mucosae was found in dog 1. Dogs 1 and 2 recovered well and vomiting ceased. Dog 3 continued to vomit because of a pyloric stenosis, mainly due to muscular hypertrophy.     

Helicobacter pylori-induced gastritis in experimentally infected conventional piglets.

A conventional nonmutant animal that could be experimentally infected with Helicobacter pylori isolates would be a useful animal model for human H. pylori-associated gastritis. Gnotobiotic and barrier-born pigs are susceptible to H. pylori infection, but attempts to infect conventional pigs with this bacterium have been unsuccessful. In the present study, a litter of eight 20-day-old crossbreed piglets were purchased from a commercial farm. Six of them were orally challenged two to five times at different ages, between 29 and 49 days, with doses of H. pylori inoculum containing approximately 10(9) bacterial cells. Two animals served as controls. The inoculation program began 2 days postweaning when the piglets were 29 days of age. Prior to every inoculation, the piglets were fasted and pretreated with cimetidine, and prior to the first and second inoculation each piglet also was pretreated with dexamethasone. The challenged piglets were euthanasized between 36 and 76 days of age. H. pylori colonized all six inoculated piglets. The pathology of the experimentally induced gastritis was examined macroscopically and by light and electron microscopy. H. pylori induced a severe lymphocytic gastritis in the conventional piglets and reproduced the large majority of the pathologic features of the human disease. Therefore, the conventional piglet represents a promising new model for study of the various pathogenic mechanisms involved in the development of lesions of the human H. pylori-associated gastritis.     

Multiple polyps of the gastric mucosa in two dogs

Multiple polyps of the gastric mucosa are described in a 6-year-old male Airedale and a 6-year-old male French Bulldog. The first dog suffered from chronic vomiting while the second vomited for only 5 days and exhibited signs of pyloric stenosis. Gastroscopic examination revealed broad, thick rolls in the Airedale's antral mucosa and radiographically the antral wall was rigid and normal peristalsis was absent. Radiographic examination of the French Bulldog revealed pyloric stenosis without passage of contrast material through the pyloric canal.
In biopsy specimens from the Airedale there were long thin foveolae and slight cellular infiltrations. Multiple polyps of the gastric mucosa were found throughout the antrum at necropsy. The resected antrum of the French Bulldog had a villous growth pattern with adenomatous formations and inflammation.     

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

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 Steiner-stained 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.     

Diversity in bacterium-host interactions within the species Helicobacter heilmannii sensu stricto

Helicobacter (H.) heilmannii sensu stricto (s.s.) is a zoonotic bacterium that naturally colonizes the stomach of dogs and cats. In humans, this microorganism has been associated with gastritis, peptic ulcer disease and mucosa associated lymphoid tissue (MALT) lymphoma. Little information is available about the pathogenesis of H. heilmannii s.s. infections in humans and it is unknown whether differences in virulence exist within this species. Therefore, a Mongolian gerbil model was used to study bacterium-host interactions of 9 H. heilmannii s.s. strains. The colonization ability of the strains, the intensity of gastritis and gene expression of various inflammatory cytokines in the stomach were determined at 9 weeks after experimental infection. The induction of an antrum-dominant chronic active gastritis with formation of lymphocytic aggregates was shown for 7 strains. High-level antral colonization was seen for 4 strains, while colonization of 4 other strains was more restricted and one strain was not detected in the stomach at 9 weeks post infection. All strains inducing a chronic active gastritis caused an up-regulation of the pro-inflammatory cytokine IL-1β in the antrum. A reduced antral expression of H⁺/K⁺ ATPase was seen in the stomach after infection with 3 highly colonizing strains and 2 highly colonizing strains caused an increased gastrin expression in the fundus. In none of the H. heilmannii s.s.-infected groups, IFN-γ expression was up-regulated. This study demonstrates diversity in bacterium-host interactions within the species H. heilmannii s.s. and that the pathogenesis of gastric infections with this microorganism is not identical to that of an H. pylori infection.