Helicobacter mustelae-induced Gastritis and Elevated Gastric ph in the Ferret (Mustela putorius furo)

INFECTION AND IMMUNITY, June 1991, p. 1875-1880 0019-9567/91/061875-06$02.00/0 Copyright 1991, American Society for Microbiology Vol. 59, No. 6 Helicobacter mustelae-induced Gastritis and Elevated Gastric ph in the Ferret (Mustela putorius furo) JAMES G. FOX,* GLEN OTTO,t NANCY S. TAYLOR, WILLIAM ROSENBLAD, AND JAMES C. MURPHY Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received 28 December 1990/Accepted 6 March 1991 Helicobacter mustelae has been cultured from the stomachs of ferrets with chronic gastritis; the lesions in the stomach have many of the same histological features seen in H. pyloni gastritis in humans. To determine whether H. mustelae-negative ferrets with normal gastric mucosa were susceptible to colonization and whether gastritis developed after infection, four H. mustelae-negative ferrets treated with cimetidine were inoculated orally on two successive days with 3 ml (1.5 x 10O CFU) of H. mustelae; eight age-matched H. mustelae-negative ferrets served as controls. All four ferrets became colonized; H. mustelae persisted through week 24 of the study, as determined by positive gastric culture, tissue urease, and Warthin-Starry staining of gastric tissue. Superficial gastritis developed in the oxyntic gastric mucosa, and a full-thickness gastritis, composed primarily of lymphocytes and plasma cells plus small numbers of neutrophils and eosinophils, was present in the antrum. The inflammation was accompanied by an elevation of immunoglobulin G antibody to H. mustelae. At 4 weeks post-inoculation, the four infected (experimental) ferrets developed an elevated gastric ph (4.0 to 5.2) for 2 weeks. The eight control ferrets did not have gastritis; H. mustelae could not be demonstrated in gastric tissue via culture, nor was there an immune response to the bacteria. In ferrets, H. mustelae readily colonizes the stomach and produces a gastritis, a significant immune response, and, like H. pyloni infection in humans, a transient elevated gastric ph after Helicobacter infection. The number of species in the genus Helicobacter (previously designated Campylobacter) has expanded since the original description by Marshall and Warren of H. pyloriassociated gastritis in humans (15, 26). New additions to the genus include H. mustelae, isolated from the stomachs of ferrets, and H. felis, isolated from gastric mucosa of the cat and dog (10, 13, 21). Unfortunately, in terms of animal model development to study pathogenesis, H. pylori appears to have a limited host range; this species naturally colonizes humans and nonhuman primates (2, 26). Experimentally, H. pylori will infect gnotobiotic pigs and dogs but does not infect rodents, lagomorphs, or ferrets (3, 7, 14b, 19). We have demonstrated that H. felis will cause an active chronic gastritis when inoculated orally into gnotobiotic mice and rats (14a, 20). Natural infection of dogs with H. felis and a second gastric organism called "Gastrospirillum hominis" (not grown in artificial medium) is associated with lymphoreticular hyperplasia, dilation of parietal cell caniliculi, and focal degeneration of parietal cells (18, 22). Natural H. mustelae infection in the ferret is associated with chronic transmucosal inflammation in the distal antrum. This mimics the diffuse antral gastritis of H. pylori described in some humans but does not replicate the severe degree of active gastritis seen in many cases of H. pylori gastritis in humans. Also, in our experience, every ferret we have found with chronic gastritis was infected with H. mustelae, while specific-pathogen-free (SPF) ferrets not infected with H. mustelae do not have gastritis. Another intriguing feature of H. pylori gastritis in humans is the development of hypochlorhydria and the presence of H. pylori in parietal cells of infected patients (1, 5, 16). This study was designed to ascertain whether H. mustelae * Corresponding author. t Present address: Animal Resource Center, University of Chicago, Box 144, Chicago, IL 60637. 1875 administered orally to SPF ferrets known to be free of the gastric organism and refractory to H. pylori infection would produce a gastritis similar to that noted in ferrets naturally infected with H. mustelae and whether colonization of the stomach with H. mustelae results in elevation of fasting gastric ph. MATERIALS AND METHODS Animals. Twelve ferrets aged 13 months were monitored for a period of 12 months to ascertain that they were negative for H. mustelae. To establish that the ferrets were H. mustelae negative, biopsies of the gastric antrum were taken from each ferret at 3, 4, 5, 8, and 11 months of age. These biopsy samples were cultured for H. mustelae, assayed for urease, and examined histologically for gastritis and H. mustelae by Warthin-Starry stain. Serum samples from each ferret were analyzed for immunoglobulin G (IgG) antibody to H. mustelae at 5 and 8 months of age. All 12 ferrets were negative for H. mustelae by this battery of tests at all time points. Previously, when these ferrets were 6 weeks of age, four of them had been experimentally infected with H. pylori; the other eight ferrets had served as negative controls. None of the four ferrets experimentally infected with H. pylori became infected, as judged by culture, tissue urease, histology, and enzyme-linked immunosorbent assay (ELISA) for H. pylori (14b). When the ferrets reached 13 months of age, the four ferrets previously challenged with H. pylori were experimentally dosed with H. mustelae, and the other eight ferrets served as controls. Ferrets were housed in animal facilities accredited by the American Association for Accreditation of Laboratory Animal Care for the 12 months prior to and during the study. Adult ferrets were maintained in singly suspended stainless steel cages (13 by 24 by 17 in. [ca. 33 by 61 by 43 cm]). Ferrets were given food (Purina Cat Chow; Ralston Purina Co., St. Louis, Mo.) and water ad libitum. To minimize the

1876 FOX ET AL. INFECT. IMMUN. TABLE 1. Experimental H. mustelae infection in ferrets Culture and urease assay resultsa at time p.i.: Ferret 1 wk 2 wk 4 wk 8 wk 12 wk 24 wk no. c u C U C U C U C U C U A F A F A F A F A F A F A F A F A F A F A F A F 1 + + - (21) - (21) + + - (24) + (3) + + + (4) + (1) + + + (2) + (2) + + + (1) + (6) + + + (7) + (2) 2 - - -(20) -(20) + - + (1.5) - (24) + + + (4) + (1.5) + + + (4) + (2) + + + (1) + (4) + + + (4) NDb 3 + + (16) + (8) + + + (5) + (2) + + + (4) + (1) + + + (6) + (2) + + + (2) + (8) + + + (4) + (2) 4 + + -(8) ± (15) + + - (24) + (6) + + + (4) + (4) + + + (1.5) ± (7) + + + (6) + (1) + + + (3) + (2) a C, culture for H. mustelae; U, urease production; A, antrum samples; F, fundus samples. Numbers in parentheses indicate hours of tissue incubation for the urease reaction. b ND, not done. potential for cross-contamination during the course of the experiment, H. mustelae-infected and control ferrets were housed in different buildings. Bacterial inocula. H. mustelae 89-977-1683, isolated from a ferret with gastritis, was used for oral dosing. It was not frozen prior to inoculation except as noted below. The organism was isolated and, on first passage, was grown for 72 h at 37 C under microaerophilic conditions on 5% lysed horse blood agar. The bacteria were harvested from the blood agar and suspended in 30 ml of brain-heart infusion broth with 30% glycerol. Half was used for dosing on day 1 of the experiment, and the other half was frozen and stored at -700F. Experimental design. Four SPF ferrets were fasted overnight, weighed, and given cimetidine (10 mg/kg) intramuscularly. After 1 h, ferrets were anesthetized with ketamine and acepromazine (2.5 and 0.25 mg/kg, respectively) intramuscularly. Three milliliters of broth containing 1.5 x 108 CFU of H. mustelae per ml was inoculated into the stomach, followed by 1 ml of broth to ensure that the full inoculum was delivered into the stomach. On day 2 of the experiment, the remaining half of the inoculum was thawed (bacteria were assessed for viability by phase microscopy), and the ferrets were given a second equal dose of H. mustelae following the protocol outlined for day 1. At 1, 2, 4, 8, 12, and 24 weeks postinfection (p.i.), biopsy samples (for culture, urease assay, and histology) were obtained via gastroscope from the antrum and body (three from each site) of anesthetized ferrets. Similar biopsy samples were obtained from the eight age-matched controls at 8 and 12 weeks of study. Urease mapping test. Antral and fundic biopsy specimens from both infected and control ferrets at each time point were assayed for urease production by the method of Hazell et al. (17). Microbiology. Stomach biopsy samples were processed within 1 h, homogenized with a sterile tissue grinder, and inoculated onto blood agar plates supplemented with trimethoprim, vancomycin, and polymyxin B (Remel, Lenexa, Kans.). The plates were incubated at 37 C in microaerophilic conditions in vented jars containing N2, H2, and CO2 (80:10: 10) for 3 to 7 days. Bacteria were identified as H. mustelae by Gram's stain, morphology, strong oxidase, catalase, and urease positivity, sensitivity to nalidixic acid, and resistance to cephalothin (10, 13). Gastric ph. Gastric ph was monitored in four ferrets during the two days of dosing and on days 7, 16, 34, 38, 49, 56, 62, 94, and 162 post inoculation. The eight control ferrets had gastric ph measured on days 50, 57, and 99 of the study. A pediatric ph probe (Synectics SYN-02 ph meter; Synectics Medical, Inc., Irving, Tex.) was used. Before the ph probe was placed, ferrets were anesthetized with ketamine and acepromazine at the same doses used when administering the inoculum. On days 7, 16, 34, 62, 94, 99, and 162, ferrets were also given atropine intramuscularly (0.04 mg/ kg); on days 0, 1, 38, 49, 50, 56, and 57, atropine wasn't given. Histopathologic examination. Antral and fundic biopsy samples for histopathologic examination were taken adjacent to samples for bacterial culture and tissue urease. Tissue were fixed in neutral buffered 10% Formalin, processed by standard methods, and embedded in paraffin; sections were cut 5,um thick and stained with hematoxylin and eosin (H&E) and by the Warthin-Starry method. A pathologist without foreknowledge of the source of the biopsy specimens examined the sections for histological changes and presence of H. mustelae. ELISA for H. mustelae antibody. Antigen was prepared from whole-cell extracts by previously published methods for H. mustelae ELISA used in our laboratory (11, 12). Three isolates of H. mustelae (including ATCC 43772) from gastric biopsy samples of ferrets were used. The ELISA was carried out by the methods of Fox et al. (11, 12). The titer of serum samples was expressed as the dilution of serum giving a reading equal to the mean plus 2 standard deviations (SD) of the negative control values. Sera. Sera from H. mustelae-infected animals was collected at day 0 and 1, 2, 4, 8, 12, 14, and 24 weeks; control sera from the eight SPF ferrets were taken at 8 and 12 weeks. RESULTS Colonization of the stomach with H. mustelae. The culture and urease results from biopsy specimens of gastric antrum and fundus indicate that experimentally infected ferrets were colonized with H. mustelae by 2 weeks p.i. Positive urease test results on antral and fundic tissue correlated well with the positive H. mustelae cultures; the rapid urease reaction (within 2 h) in many biopsy samples indicated heavy colonization (17) (Table 1). Warthin-Starry staining of stomach samples also indicated colonization with H. mustelae in both the antrum and fundus of all ferrets sampled on weeks 2 through 24. Figure 1 depicts H. mustelae in the stomach of an infected ferret. Short rod-shaped to slightly curved bacteria typical of H. mustelae were present on the surface of the gastric epithelium within the mucus layer and within gastric pits. ELISA. At 2 weeks after H. mustelae inoculation, the four

VOL. 59, 1991 H. MUSTELAE INDUCES GASTRITIS IN FERRETS 1877 GL-,-.w, e.. Jqfzw~ a.00 _ FIG. 1. Large numbers of bacteria (arrowheads) colonizing the lumen of a fundic gland in the stomach of a ferret orally inoculated with H. mustelae. Warthin-Starry stain. Magnification x750. H. mustelae-infected ferrets had a fourfold increase in H. mustelae IgG antibody titer over baseline H. mustelae antibody titers (<1:64) for the 12 ferrets (four experimental and eight control) (Fig. 2). ELISA values on subsequent analysis, during the 24 weeks of the study, indicated a steady increase in H. mustelae IgG antibody titers in the four H. mustelae-infected ferrets, whereas H. mustelae antibody titers in the eight control ferrets remained <1:64. Measurement of gastric ph. Cimetidine was clearly effective in producing an apparent hypochlorhydria (as reflected by elevated gastric ph) for the oral inoculation of H. mustelae on two successive days (Fig. 3). At weeks 1 and 2 p.i., gastric ph remained acidic in all four ferrets. However, on week 4 p.i., one of the ferrets had a gastric ph of 4.7; the ph measured 4 days later in all four ferrets was elevated (ph 4.0 to 5.2). The raised gastric ph was transient (approximately 2 weeks), as evidenced by a return to acidic ph in all ferrets when measured 1, 2, and 4 weeks after the period of apparent hypochlorhydria. Control ferrets had acidic gastric ph values on days 50, 57, and 99 of the study (Fig. 2). Atropine had no appreciable effect on gastric ph. Histopathology. Biopsy specimens from the eight control ferrets taken during weeks 8 and 12 of the study were conspicuous by the uniform absence of any significant inflammatory cell infiltration (Fig. 4). Minimal numbers of lymphocytes and occasional polymorphonuclear leukocytes were scattered throughout the subglandular portions of both the fundic and pyloric mucosa. Biopsy specimens from the four H. mustelae-infected ferrets obtained at 1, 2, 4, 8, 12, and 24 weeks p.i. had focal, relatively mild inflammatory cell infiltrates in the pyloric mucosa of three of the four ferrets, whereas the fundic mucosa were similar to those from the controls except for the presence of a focal superficial gastritis consisting of lymphocytes and occasional neutrophils. The inflammatory in. 0 1:100 00 1:1000 / N 6- P 4- -1-i -0--- 2 -h- 3 - - 4 I 1:100 < 1:64 1' Control 2 4 8 12 14 24 Week FIG. 2. H. mustelae IgG titers in four experimentally infected ferrets. Each symbol represents an individual ferret: L, ferret 1; 0, ferret 2;., ferret 3; *, ferret 4. 2- I I I I I I * 0 6 1 7 1 6 34 38 49 56 62 94 162 Day Post Inoculation FIG. 3. Gastric ph measurements in four ferrets experimentally infected with H. mustelae. Each symbol represents an individual ferret. Values for SPF control ferrets are also shown (x). *, Ferrets were administered H. mustelae and cimetidine on days -1 and 0.

1878 FOX ET AL. INFECT. IMMUN. 44.^ Y bsm7xt4 FIG. 4. Typical example of the minimal numbers of leukocytes FIG. 5. Example of the magnitude of the predominantly lymphoobserved in samples of the gastric mucosa of ferrets free of H. cytic infiltration of the subglandular region of the gastric mucosa mustelae. H&E. Magnification, x300. observed in samples from ferrets orally inoculated with H. mustelae. H&E. Magnification, x150. cell infiltrates in the antrum were composed primarily of lymphocytes, with a few neutrophils and eosinophils interspersed. The foci of inflammation consisted of aggregates of leukocytes primarily concentrated in the subglandular portion of the mucosa, with extension into the lamina propria toward the luminal surface and through the muscularis mucosa into the submucosa (Fig. 5 and 6). A few cystic glands were observed in selected pyloric biopsy samples, but this was an inconsistent finding. From the biopsy samples evaluated, there was no clearly defined evidence to suggest a significant increase in the severity of lesions during the 6-month interval of the experiment. DISCUSSION Arguments supporting the contention that H. pylori is a pathogen are based on several interrelated observations. Warren and Marshall's initial discovery that H. pylori was associated with gastritis was supported by further studies showing that in patients with H. pylori gastritis treated with antimicrobial agents, the gastritis resolved (30). The fulfillment of Koch's postulate with two human volunteers who ingested H. pylori and developed gastritis also favors the role of H. pylori as a gastric pathogen (23, 28). More recently, H. pylori has been associated with the development of duodenal ulcers (25, 33, 35). Efforts to study the pathogenesis of H. pylori in animal models have been hampered by the organism's host specificity. To date the only reliable model for studying H. pylori infection has been the gnotobiotic pig (19). A recent report suggests that the gnotobiotic dog and SPF pig (raised under barrier conditions) may also be appropriate models for some H. pylori pathogenesis studies (7, 31). Preliminary evidence indicates that nonhuman primates may also be susceptible to H. pylori infection (2). Unfortunately, many of these species are also colonized with a nonculturable gastric spirillum, Gastrospirillum hominis, which also occasionally infects humans (14, 27, 34). In addition, these models have disadvantages in regard to availability, housing, cost, and ease of administering antimicrobial agents for eradication studies. Though the rat and mouse are not colonized by H. pylori, we have shown that H. felis colonizes and produces a gastritis in both gnotobiotic rats and mice (14a, 20). These rodent models will afford the opportunity to study Helicobacter gastric infection, but because of their size, the taking of repeated gastric biopsy samples from individual animals is not feasible. The ferret offers several advantages for the study of Helicobacter-induced gastritis. The ferret has a stomach with anatomical and physiological characteristics similar to those of humans; it is readily available commercially, is easy to maintain in a research setting, and has a tractable dispo-

VOL. 59, 1991 FIG. 6. High magnification of a sample of the gastric mucosa from a ferret orally inoculated with H. mustelae reveals leukocytes (arrowheads) infiltrating the lamina propa. H&E Magnification, x 300. sition (8). Another attractive feature of this model is that the ferret stomach is naturally colonized by H. mustelae, which is closely related phenotypically, by DNA homology and RNA sequencing data, and biochemically to H. pylori (9, 10, 13). H. mustelae, like H. pylori, closely adheres to gastric mucosa and produces a gastritis similar to that noted in children and some adults infected with H. pylori (6, 12); also we have recently found that H. mustelae has urease subunits similar to those reported for H. pylori (6a, 11). These similarities are important because both adherence and urease production have been suggested to play a role in the pathogenesis of H. pylori gastroduodenal disease. Our original studies indicated that H. mustelae colonizes nearly 100% of the ferrets we have sampled and that colonization occurs shortly after weaning. The infection is accompanied by gastritis which persists in the stomach for years, probably for the life of the animal (9, 11). This prompted us to derive and maintain a small nucleus of SPF ferrets without H. mustelae infection. Also, therapy studies indicate that eradication of H. mustelae is possible by using antimicrobial agent regimens similar to those used to eradicate H. pylori in humans (29). The present study fulfills Koch's postulates and demonstrates that H. mustelae is a gastric pathogen. The organism, inoculated into previously H. mustelae-negative ferrets, H. MUSTELAE INDUCES GASTRITIS IN FERRETS 1879 readily colonized the gastric mucosa and produced an immune response and chronic gastritis, all features which are noted in ferrets naturally colonized with H. mustelae (11, 13). The stomach lesions, consisting of focal chronic antral gastritis induced by the experimental infection with H. mustelae, are similar to the gastritis observed in young ferrets that have been naturally colonized with the organism for a limited time period (14b). The focal nature of the gastritis may explain the lack of significant inflammatory response in one of the infected ferrets. Another intriguing finding in the current study was the apparent transient hypochlorhydria observed approximately 4 weeks after the infection. According to the urease tissue assay, this period coincided with heavy H. mustelae colonization of the fundus. In the experimentally infected ferrets over time (>8 weeks), the organism, as in most naturally infected ferrets, colonizes the antrum in greater numbers than the fundus (11). Of the two humans who voluntarily ingested H. pylori, one had confirmed elevated gastric ph lasting several weeks (28). The previous observation of epidemic hypochlorhydria in volunteer patients undergoing gastric physiology studies is now thought to be related to acute infection with H. pylori (32). latrogenic acute H. pylori gastritis in another patient was accompanied by a similar episode of achlorhydria noted during the second week of infection and lasting approximately 2 months (16). The mechanism which causes the transient gastric hypochlorhyria observed in the H. mustelae-infected ferrets and in human cases of H. pylori gastritis is unknown. Marshall et al. recently demonstrated in vitro that H. pylori is able to utilize physiological concentrations of urea sufficient to neutralize the hydrogen ions present in gastric juice (24). They suggested that the ph neutralization occurring during H. pylori metabolism of urea via the action of urease may have been sufficient to explain the basal hypochlorhydria seen in the volunteer experiment described previously (24, 32). They theorized that the parietal cell failure may be due to toxic effects of the ammonia production occurring during urea utilization by H. pylori (24). Others have shown, by using in vitro suspensions of rabbit parietal cells, that H. pylori can switch off parietal cells; this suggests that a nondialyzable protein is responsible for the antisecretory effect on the parietal cell (4). We have recently demonstrated that H. mustelae switches off both ferret and rabbit parietal cells in vitro (34a). The possibility of direct inhibitory action on parietal cells is further supported by the observation of gastric spirilla in close association with parietal cells in humans infected with H. pylori, in dogs, cats, and nonhuman primates infected with H. felis or Gastrospirillum hominis, and in rodents experimentally infected with H. felis (5, 18, 19a, 22, 34). In dogs, in which spiral organisms in parietal cells have been noted frequently, the parietal cell caniculi were sometimes dilated, and occasionally parietal cells were vacuolated and degenerated (18). Further studies are needed to prove or disprove the proposed theories regarding the pathogenesis of Helicobacter-associated hypochlorhydria. In conclusion, H. mustelae infection in the ferret has many of the same features observed in H. pylori-associated gastritis in humans. Our observations concerning naturally occurring H. mustelae gastritis in ferrets have been reproduced by experimental infection with H. mustelae in SPF ferrets. The ferrets developed an infection which resulted in a persistent, chronic gastritis and significant immune response. The additional feature of elevated gastric ph in H. mustelae-infected ferrets further supports the use of this

1880 FOX ET AL. model to study the natural progression and pathogenesis of Helicobacter-induced gastritis in mammalian hosts. ACKNOWLEDGMENTS This research was supported in part by Public Health Service grants R01-A125631 from the National Institute of Allergy and Infectious Diseases, P01-CA-26731 from the National Cancer Institute, and RR01046 and T32-RR07036 from the Center for Research Resources. REFERENCES 1. Barthel, J. S., T. V. Westblom, A. D. Havey, F. Gonzalez, and E. D. Everett. 1988. Gastritis and Campylobacter pylori in healthy asymptomatic volunteers. Arch. Intern. Med. 148:1149-1151. 2. Baskerville, A., and D. B. Newell. 1988. Naturally occurring chronic gastritis and C. pylori infection in the rhesus monkey: a potential model for gastritis in man. Gut 29:465-472. 3. Cantorna, M. T., and E. Balish. 1990. Inability of human clinical strains of Helicobacter pylori to colonize the alimentary tract of germ-free rodents. Can. J. Microbiol. 36:237-241. 4. Cave, D. R., and M. 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