Helicobacter-like organisms: Histopathological examination of gastric biopsies from dogs and cats

J. Comp. Path. 1995 Vol. 112, 307-318

Helicobacter-like Organisms: Histopathological

Examination of Gastric Biopsies from Dogs and Cats W. Hex~manns, K. Kregel, W. Breuer and J. Lechner* Institute of Veterinary Pathology, University of Munich, Veteriniirstrasse 13, D-80539 Miinehen, Germany and *Clinic for Small Animals, Wertachstrasse 1, D-90451 Niirnberg, Germany

Summary Gastric biopsies from the fundic gland region of 122 dogs and 127 cats were subjected to histopathological examination. The aim of the study was to determine infection rates and degrees of colonization by Helicobacter-like organisms (HLOs), and to ascertain their possible relationship to histopathological changes. In all, 82% of the dogs and 76% of the cats had an HLO infection. The most striking histopathological changes were glandular degeneration with accumulation oflymphocytes and neutrophilic granulocytes (dogs, 21%; cats, 39%), fibrosis of the lamina propria mucosae (dogs, 41%; cats, 58%), oedema in the lamina propria mucosae (dogs, 54%; cats, 23%), lymphoid follicles (dogs, 17 %; cats, 19 %) and lymphoplasmacytic infiltrates. A relation between the degree of colonization by HLOs and the extent of histopathological changes could only be discovered in the cats. It was not possible to ascertain whether these bacteria, irrespective of the degree of colonization, were responsible for the histopathological changes in the dogs.

Introduction T h e presence of spiral-shaped bacteria in the stomach was originally described by Rappin (1881). Since then, these microorganisms--initially called spirillae or spirochaetes, then Campylobacter-like organisms, and now Helicobacter-like organisms (HLOs)--have regularly been mentioned in connection with gastric examinations, sometimes in conjunction with histopathological lesions. The HLOs are a group of Gram-negative, spiral-shaped bacteria, equipped with flagella, which can be subdivided into gastric and nongastric species (Lee and O'Rourke, 1993). Gastric Helicobacter spp. are urease-positive and have been described in m a n y animal species, including dogs and cats (Salomon, 1896; Weber et al., 1958; Lockard and Boler, 1970; Henry et al., 1987), ferrets (Fox et al., 1986, 1991a, b), pigs (Mendes et al., 1990, 1991), calves (Ganther and Schulze, 1992), cheetahs (Eaton et al., 1991, 1993a, b) and various species of monkey (Doenges, 1939; Sato and Takeuchi, 1982; Curry et al., 1987; Newell et a/.,1987), and also in m a n (Warren and Marshall, 1983). Ever since they were mentioned by Warren and Marshall (1983) in connection with h u m a n type-B gastritis, renewed interest has been taken in these microorganisms. Helicobacter pylori, the species occurring in man, has also been linked with gastric ulcers, duodenal ulcers and adenocarcinoma of the stomach (Rautelin and Kosunen, 1991). 0021-9975/95/030307 + 12 $08.00/0

9 1995 Academic Press Limited

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Dogs and cats have been shown to be infected with several species of Helicobacter (Salomon, 1896; W e b e r et al., 1958; W e b e r and Schmittdiel, 1962; Lockard and Boler, 1970). H. felis, a species first isolated in cats but also occurring in dogs, has been definitively classified (Lee et al., 1988). Gastrospirillum hominis, another H L O , has so far not been cultivated in artificial media (Solnick et al., 1993) but is said to be the most commonly occurring H L O of cats and dogs (Lockard and Boler, 1970; Lee et al., 1988; Lee and O'Rourke, 1993). M a n y investigators cite a 100% rate of H L O infection (Salomon, 1896; W e b e r et al., 1958; H e n r y et al., 1987), the earliest age of infection being the 4th week of life (Salomon, 1896), although Weber et al. (1958) failed to detect such bacteria in 1-month-old kittens. The manner in which the infection is transmitted in animals and in man has not yet been clarified, but oral transmission is assumed (Lee et al., 1991). Gastric H L O s are found in the cardiac, fundic and pyloric gland regions. According to Oshikawa (1923), they only occur in glands endowed with parietal cells. H L O s demonstrate considerable affinity for parietal cells and are found in their intracellular canaliculi as well as in the cytoplasm; they have, however, also been observed in large numbers in the mucus covering the surface epithelium, the gastric pits and the glandular lumina (Salomon, 1896; Weber et al., 1958; H e n r y et al., 1987). Clinical symptoms are not usually observed in HLO-infected animals. In many studies, such animals were described as clinically healthy, and had usually been humanely killed for other reasons (e.g. Bizzozero, 1893; Salomon, 1896; Regaud, 1909; Kolmer and Wagner, 1916; Oshikawa, 1923; W e b e r et al., 1958; H e n r y et al., 1987). The aim of this study, based on an examination of gastric biopsies, was to determine H L O infection rates and degrees of colonization in cats and dogs, to record histopathological lesions, and to ascertain whether there was any correlation between H L O occurrence and morphological lesions. Materials and Methods

Specimens Gastric biopsies from 122 dogs and 127 cats were subjected to histopathological examination; these biopsies had been submitted between 1990 and 1993 for routine diagnosis. Of the 122 dogs, 56 were female, 16 of the latter castrated; four of the 66 male dogs were castrated. The mean age was 5"5 years (range 0-5 to 14 years). Of the 127 cats, 41 were females, 18 of the latter castrated; of the 86 male cats, 65 were castrated. The mean age was 6"9 years (range 2 months to 16 years). A small number of the biopsies came from animals classified as having no gastric disorders, and some came from animals suffering from secondary gastric diseases such as uraemic gastropathy. The majority of the animals biopsied presented gastrointestinal symptoms that were not attributable to diseases in other organs such as the liver, kidneys, pancreas and heart. No antibacterial treatment had been administered before biopsy, but a change of diet or the administration of antacids may sometimes have taken place. Details of age, sex, clinical symptoms and endoscopic results were available for most of the animals. Collection and Processing of Biopsies Food was usually withheld for 12 h before endoscopy and biopsy. The dogs were anaesthetized with droperidol, fentanyl and etomidat, the cats with propofol. The

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O r g a n i s m s in D o g s and C a t s

Table 1 C l i n i c a l s y m p t o m s and endoscopic findings in the dogs and c a t s f r o m which biopsies w e r e examined

Number of animals with the various clinical symptoms and endoscopicfindings Observations

Dogs (n = 122)

Cats (n = 127)

16 20 70 36 11 1 2 1

31 11 65 34 14 7 3 3

18 28 13 5 2

12 12 8 12 4

Clinical symptoms None No record Vomiting Diarrhoea Loss of appetite Emaciation Fever Polyphagia Endoscopic findings Duodenal reflux Reddening of mucosa Oedema Erosions Ulcers

biopsy was performed with the help of a flexible gastroscope under visual control, the aim being to remove lesions such as erosions, the periphery of ulcers and lymphoid follicles. The tissue samples were fixed in 10% buffered formalin. The biopsies were embedded in glycol methacrylate (Hermanns et al., 1981) and sections 2 gm thick were prepared and stained with haematoxylin and eosin (HE) and by the GiemsaWright method.

Evaluation of Histopathological Changes and HLO Infection The dog and cat biopsies were evaluated separately. Generally, several biopsies were available from each animal. The specimens used all came from the fundic gland region, the biopsy evaluated being in each case that with the severest lesions. Histopathological observations were made on the various parts of the gastric mucosa: gastric pits, lumina of the glands, surface epithelium, glandular epithelial cells, and interglandular connective tissue. The frequency of occurrence of free cells in the connective tissue, such as lymphocytes, plasma cells, neutrophilic granulocytes, eosinophilic granulocytes, mast cells, macrophages, pigment cells, and globular leucocytes was graded (see legend to Table 2). In addition, the degree of HLO colonization in the various parts of the gastric mucosa was quantified according to the Sydney system (Price, 1991), frequently used for the evaluation of gastric biopsies in man. Correlation was attempted between the degree of HLO colonization and presence of lymphocytes, plasma cells, neutrophilic granulocytes, lymphoid follicles, glandular degeneration and surface epithelial changes. For this purpose, the animals were divided into groups according to the extent of HLO colonization, i.e. non-existent, slight, medium-grade or high-grade. These groups could then be set against the mean "grades" of the various histopathological observations.

Results T h e c o m m o n e s t clinical symptoms reported were vomiting a n d d i a r r h o e a (see Table 1). Lack of appetite, loss of weight, a n d less frequently fever a n d

310

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et

al.

polyphagia, had also been noted. Endoscopic examination frequently revealed duodenal reflux and reddening of the mucosa, and occasionally oedema, isolated erosions and ulcers. HLOs were found in the biopsies of 100 dogs (82%) and 96 cats (76%), being localized in the mucus covering the surface epithelium, the gastric pits, the glandular tumina and the parietal cells (Figs 1 and 2). The question of whether HLOs also occur in the chief cells and mucous neck cells could not be answered definitively by light microscopy. No HLOs were observed in the surface epithelial cells, the endocrine cells or the connective tissue. The bacteria were often found in large clusters in the gastric pits, but they also occurred in isolation; normally they were arranged vertically to the lumen of the stomach. Colonization of the gastric glands occurred mainly in their upper parts, i.e. close to the lumen; due to the narrowness of the glandular lumina, the H L O were generally arranged end-to-end in this site, but they sometimes occurred in clusters that completely occupied the lumen. Generally the parietal cells contained only isolated bacteria, but sometimes the organisms were present in such large numbers that they occupied almost the entire cell. T h e y were usually found in vacuoles which may have been either cytoplasmic vacuoles or dilated canaliculi. A few biopsies gave the impression of bacteria in a state of breakdown and lysis. The histopathological lesions and the frequency of their occurrence are given in Table 2. The mucus content of the surface epithelial cells was clearly reduced in some cases, giving the cells a cuboid shape. Isolated intraepithelial lymphocytes were frequently observed; in some cases, however, clusters of cells were found within the surface epithelium. Globular leucocytes were seldom observed in cats, but they were occasionally seen between the epithelial cells, particuIarly in the region of the gastric pits. Isolated goblet cells were occasionally observed, but erosions or ulcers were not seen. The breadth and depth of the gastric pits varied; in some cases they were very flat and wide or had even disappeared altogether. Dilatation of the lumina of the fundic glands was observed occasionally, cysts rarely. Metaplasia of the glandular epithelium could not be definitively identified; as the biopsy sites were not known, the samples may have come from the area adjacent to the pyloric gland region. Atrophy of the mucous membrane could not be appraised in most cases, as m a n y of the biopsies did not include the lamina muscularis mucosae. Occasionally, entire glandular segments were necrotic. In some degenerating glands, increased numbers of lymphocytes, occasionally even neutrophilic granulocytes, were detected (Fig. 3). Other glandular segments, on the other hand, appeared to be strangulated by surrounding connective tissue, without any clear evidence of the presence of inflammatory cells (Fig. 4). O f the glandular epithelial cells, only the parietal ceils were markedly abnormal. Vacuoles were found in a relatively high proportion of the parietal cells of all biopsies, some in the juxtanuclear position, others at the margin of the cell. Some parietal cells appeared larger than others, reaching approximately twice the average parietal-cell size. In a very few cases, cell nuclei

Helicobacter-like Organisms in Dogs and Cats

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Fig. 1.

Helicobacter-like organisms (HLOs) in the gastric pits of the fundic gland region of a 13-year-old cat. Giemsa. Bar = 25 gin.

Fig. 2.

H L O s in the glandular lumina and parietal cells (arrow) of the same cat as in Fig. 1. Giemsa. Bar = 25 gm.

in the process of degeneration, in the form of karyolysis or even karyorrhexis, were observed. Changes in the connective tissue of the lamina propria, in particular fibrosis, were frequently observed, both focally and diffusely (Fig. 5). Hypertrophy of

312

W. Herm~nns et aL Table 2 Results o f histological e x a m i n a t i o n o f gastric b i o p s i e s obtained from dogs and cats Number of animals with the various histopathologicalfindings

Histopathologicalfindings Helicobacter-like organisms Surface epithelial cells Reduction of mucus content Cuboid shape of cells Intraepithelial lymphocytes low-grade high-grade Globular leucocytes Goblet cells Erosions/ulcers Fundic glands Dilatation Cysts Glandular degeneration single location multiple locations Free cells in the connective tissue Lymphoid follicles Lymphocytes low-grade high-grade Plasma cells low-grade high-grade Neutrophilic granulocytes low-grade high-grade Mast cells low-grade high-grade Eosinophilic granulocytes low-grade high-grade Interstitial connective tissue Fibrosis slight severe Oedema slight severe Smooth muscle hypertrophy

Dogs (n = 122)

Cats (n = 127)

100

96

38 4

50 11

45 9 0 13 0

49 27 12 12 0

13 13

3 3

14 12

22 28

21

24

79 43

77 50

71 46

79 21

102 10

101 3

92 20

68 49

12 0

12 0

38 12

49 25

51 15 10

28 1 13

Microscopical grading, which was made at • 400 magnification, represents the mean values obtained from at least three biopsy sites (Lymphocytes: low grade < 10 cells/field; high-grade > 10 cells/field. Plasma cells, neutrophilic granulocytes, eosinophilic granulocytes, mast cells: low-grade <5 cells/field; high-grade >5 cells/field.)

the smooth muscle fibres surrounding the glands in the region of the lamina propria in particular was occasionally observed, with several smooth muscle fibres extending like pillars into the region between the gastric pits. Various free cells were found in the lamina propria, the most frequent site

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313

Fig. 3.

Glandular degeneration with accumulation of lymphocytes and neutrophilic granulocytes, H L O s in numerous glandular lumina, and also vacuoles in the glandular epithelial cells in a 5-year-old cat. Giemsa. Bar = 25 gm.

Fig. 4.

"High-grade" fibrosis of the lamina propria with glandular degeneration but without accumulation of lymphocytes or other inflammatory cells, in a 1-year-old cat. Giemsa. Bar = 25 gm.

being the area beneath the surface epithelium. Isolated lymphocytes were found in all biopsies, being either subepithelial, diffuse or limited to the area of individual gland segments. In addition, lymphoid follicles were also found

314

W. Hermanns

et

al.

Fig. 5.

"High-grade" fibrosis of the lamina propria with isolated lymphocytes and plasma cells and also glandular degeneration (atrophic gastritis) in a 9-year-old dog. HE. Bar = 50 gin.

Fig. 6.

Lymphoid follicle in the lamina propria of the fundic gland region with "high-grade" glandular degeneration in a 6-year-old dog. Giemsa. Bar = 50 gm.

(Fig. 6). Most biopsies contained single subepithelial plasma cells and neutrophilic granulocytes; mast cells were generally localized between the glands around the blood vessels and the area between the gastric pits and the

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glands. Pigment cells, eosinophilic granulocytes and macrophages were seldom observed. Correlation between the degree of H L O colonization and the frequency of occurrence of lymphocytes and plasma cells could not be substantiated in dogs; in cats, the mean numbers of both lymphocytes and plasma cells was higher when the number of bacteria increased. In cats, increased bacterial colonization was directly related to the number of lymphoid follicles; in dogs, this was true only in cases in which H L O colonization was classified as "highgrade". Fibrosis occurred more frequently in cats, in which species it was apparently directly related to the degree of H L O colonization. There did not appear to be any link between oedema and bacterial colonization. Dogs displayed lesions of the surface epithelium most frequently in cases of mild H L O infection. In cats such lesions were associated with high-grade bacterial infection. In the presence of "high-grade" HLO colonization, glandular degeneration occurred less frequently in dogs than in cats. Discussion

Hdicobacter-like organisms were found in c. 80% of the dog and cat biopsies.

In the literature, infection rates of up to 100% are cited (Salomon, 1896; Weber et aL, 1958; Henry et al., 1987), but the earlier workers examined specimens from several gastric sites, thus increasing the probability of finding the bacteria. In addition, the dogs examined by Henry et al. (1987) had been kept in a colony, with close mutual contact. In work on H. felis in dogs, Lee et al. (1992) obtained evidence of cross-infection from an experimentally inoculated animal. Most of the dogs in our study were kept on their own, which might explain the lower rate of infection. Considerable differences in the degree of HLO colonization of the gastric mucosa of animals have been reported, and the data recorded varied according to the area of the fundic gland region biopsied. Similar findings in man led Bayerd6rffer et al. (1992) to conclude that at least four biopsies per patient should be examined. Regularly occurring histopathological lesions in the gastric biopsies were: inflammatory cells, lymphoid follicles, glandular degeneration, fibrosis and oedema. The inflammatory cells of the lamina propria consisted oflymphocytes, plasma cells, neutrophilic granulocytes and, to a small extent, eosinophilic granulocytes. Lymphoeytes could be observed in all specimens, and plasma cells and neutrophilic granulocytes in many. Whether a certain frequency of occurrence of such cells should be regarded as physiological or inflammatory is debatable. According to the German Society of Pathology (1989), the gastric mucosa should only be classed as normal if no lymphocytes, plasma cells or granulocytes can be detected in the lamina propria. The occurrence of isolated lymphocytes and plasma cells indicates minimal gastritis. On the other hand, according to the Sydney system (Price, 1991), a small number of uniformly distributed lymphocytes or plasma cells should be regarded as physiological and a single neutrophilic granulocyte per biopsy viewed as normal. In many of our biopsies, the number of lymphocytes and plasma cells was clearly

316

W. H e r m a n n s e t al.

elevated, but that of the neutrophilic and eosinophilic granulocytes rarely so. In dogs (Henry et al., 1987; Lee et al., 1992), rats and mice (Lee et al., 1990; Fox et al., 1991c) infected naturally with HLOs and artificially with H. felis, lymphoid follicles are regularly found in subglandular areas and sometimes extend between the glands. In our study, lymphoid follicles were present in 21 dogs and 23 cats, with no HLOs being found in the biopsies from three of the dogs and two of the cats. It is possible that the frequency of occurrence of lymphoid follicles as estimated on the basis of our biopsies was too low. The studies cited above on material from necropsies indicate that lymphoid follicles are generally located in areas from which biopsies are rarely taken. Glandular degeneration was generally accompanied by lymphocyte accumulation and, to a slighter degree, by the presence of neutrophilic granulocytes. In addition, glands appeared to degenerate in the absence of inflammatory cells as a consequence of high-grade fibrosis of the propria. Cats, but not dogs, demonstrated a high degree of positive correlation between glandular degeneration and degree of H L O colonization. Whether the bacteria alone caused the degeneration or whether some type of autoimmunity played an additional role was not clear. Changes that occurred both in the presence and absence of bacteria were regularly observed in the parietal cells. Changes in the parietal cells associated with H L O infection have been described by other authors (Salomon, 1896; Weber et al., 1958; Weber, 1960; Henry et al., 1987). In conclusion, the differences between the histopathological findings in dogs and cats may be summarized as follows. O e d e m a occurred more frequently and strikingly in dogs than in cats, whereas glandular degeneration and fibrosis were rarer in dogs. The correlation between degree of H L O colonization and histopathological changes in the two species also differed. In cats, but not in dogs, H L O colonization was directly related to the number of inflammatory cells, lymphoid follicles and degenerated glands and to changes in the surface epithelium. It is not clear whether these differences were related to the pathogenicity of the infecting HLOs in the two animal species, or to genetic differences in the hosts. It should be noted, however, that the histopathological changes in the dogs and cats were much less severe than those seen in H. pylori gastritis in man. References

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1

Received, Ju~ 21st, 1994 Accepted, December 2 4 th, 1994J