Vol. 59, No. 6INFECTION AND IMMUNITY, June 1991, p. 1875-18800019-9567/91/061875-06$02.00/0Copyright © 1991, American Society for Microbiology

Helicobacter mustelae-Induced Gastritis and ElevatedGastric pH in the Ferret (Mustela putorius furo)

JAMES G. FOX,* GLEN OTTO,t NANCY S. TAYLOR, WILLIAM ROSENBLAD, AND JAMES C. MURPHYDivision 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 thestomach have many of the same histological features seen in H. pyloni gastritis in humans. To determinewhether H. mustelae-negative ferrets with normal gastric mucosa were susceptible to colonization and whethergastritis developed after infection, four H. mustelae-negative ferrets treated with cimetidine were inoculatedorally on two successive days with 3 ml (1.5 x 10O CFU) ofH. mustelae; eight age-matched H. mustelae-negativeferrets served as controls. All four ferrets became colonized; H. mustelae persisted through week 24 of thestudy, 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 primarilyof 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 weekspost-inoculation, the four infected (experimental) ferrets developed an elevated gastric pH (4.0 to 5.2) for 2weeks. The eight control ferrets did not have gastritis; H. mustelae could not be demonstrated in gastric tissuevia culture, nor was there an immune response to the bacteria. In ferrets, H. mustelae readily colonizes thestomach and produces a gastritis, a significant immune response, and, like H. pyloni infection in humans, atransient elevated gastric pH after Helicobacter infection.

The number of species in the genus Helicobacter (previ-ously designated Campylobacter) has expanded since theoriginal description by Marshall and Warren of H. pylori-associated gastritis in humans (15, 26). New additions to thegenus include H. mustelae, isolated from the stomachs offerrets, and H. felis, isolated from gastric mucosa of the catand dog (10, 13, 21). Unfortunately, in terms of animal modeldevelopment to study pathogenesis, H. pylori appears tohave a limited host range; this species naturally colonizeshumans and nonhuman primates (2, 26). Experimentally, H.pylori will infect gnotobiotic pigs and dogs but does notinfect rodents, lagomorphs, or ferrets (3, 7, 14b, 19).We have demonstrated that H. felis will cause an active

chronic gastritis when inoculated orally into gnotobioticmice and rats (14a, 20). Natural infection of dogs with H.felis and a second gastric organism called "Gastrospirillumhominis" (not grown in artificial medium) is associated withlymphoreticular hyperplasia, dilation of parietal cell canili-culi, and focal degeneration of parietal cells (18, 22). NaturalH. mustelae infection in the ferret is associated with chronictransmucosal inflammation in the distal antrum. This mimicsthe diffuse antral gastritis of H. pylori described in somehumans but does not replicate the severe degree of activegastritis seen in many cases of H. pylori gastritis in humans.Also, in our experience, every ferret we have found withchronic gastritis was infected with H. mustelae, while spe-cific-pathogen-free (SPF) ferrets not infected with H. mus-telae do not have gastritis.Another intriguing feature of H. pylori gastritis in humans

is the development of hypochlorhydria and the presence ofH. 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 Chi-

cago, Box 144, Chicago, IL 60637.

administered orally to SPF ferrets known to be free of thegastric organism and refractory to H. pylori infection wouldproduce a gastritis similar to that noted in ferrets naturallyinfected with H. mustelae and whether colonization of thestomach with H. mustelae results in elevation of fastinggastric pH.

MATERIALS AND METHODS

Animals. Twelve ferrets aged 13 months were monitoredfor a period of 12 months to ascertain that they were negativefor H. mustelae. To establish that the ferrets were H.mustelae negative, biopsies of the gastric antrum were takenfrom each ferret at 3, 4, 5, 8, and 11 months of age. Thesebiopsy samples were cultured for H. mustelae, assayed forurease, and examined histologically for gastritis and H.mustelae by Warthin-Starry stain. Serum samples from eachferret were analyzed for immunoglobulin G (IgG) antibody toH. mustelae at 5 and 8 months of age. All 12 ferrets werenegative for H. mustelae by this battery of tests at all timepoints. 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 con-trols. None of the four ferrets experimentally infected withH. pylori became infected, as judged by culture, tissueurease, histology, and enzyme-linked immunosorbent assay(ELISA) for H. pylori (14b). When the ferrets reached 13months of age, the four ferrets previously challenged with H.pylori were experimentally dosed with H. mustelae, and theother eight ferrets served as controls.

Ferrets were housed in animal facilities accredited by theAmerican Association for Accreditation of Laboratory Ani-mal Care for the 12 months prior to and during the study.Adult ferrets were maintained in singly suspended stainlesssteel cages (13 by 24 by 17 in. [ca. 33 by 61 by 43 cm]).Ferrets were given food (Purina Cat Chow; Ralston PurinaCo., St. Louis, Mo.) and water ad libitum. To minimize the

1875

on October 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

1876 FOX ET AL.

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 wkno. 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) NDb3 + + (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 theexperiment, H. mustelae-infected and control ferrets werehoused in different buildings.

Bacterial inocula. H. mustelae 89-977-1683, isolated from aferret with gastritis, was used for oral dosing. It was notfrozen prior to inoculation except as noted below. Theorganism was isolated and, on first passage, was grown for72 h at 37°C under microaerophilic conditions on 5% lysedhorse blood agar. The bacteria were harvested from theblood agar and suspended in 30 ml of brain-heart infusionbroth with 30% glycerol. Half was used for dosing on day 1of the experiment, and the other half was frozen and storedat -700F.

Experimental design. Four SPF ferrets were fasted over-night, weighed, and given cimetidine (10 mg/kg) intramuscu-larly. After 1 h, ferrets were anesthetized with ketamine andacepromazine (2.5 and 0.25 mg/kg, respectively) intramus-cularly. Three milliliters of broth containing 1.5 x 108 CFUof H. mustelae per ml was inoculated into the stomach,followed by 1 ml of broth to ensure that the full inoculumwas delivered into the stomach. On day 2 of the experiment,the remaining half of the inoculum was thawed (bacteriawere assessed for viability by phase microscopy), and theferrets were given a second equal dose of H. mustelaefollowing 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) wereobtained via gastroscope from the antrum and body (threefrom each site) of anesthetized ferrets. Similar biopsy sam-ples were obtained from the eight age-matched controls at 8and 12 weeks of study.

Urease mapping test. Antral and fundic biopsy specimensfrom both infected and control ferrets at each time pointwere assayed for urease production by the method of Hazellet al. (17).

Microbiology. Stomach biopsy samples were processedwithin 1 h, homogenized with a sterile tissue grinder, andinoculated onto blood agar plates supplemented with tri-methoprim, vancomycin, and polymyxin B (Remel, Lenexa,Kans.). The plates were incubated at 37°C in microaerophilicconditions in vented jars containing N2, H2, and CO2 (80:10:10) for 3 to 7 days. Bacteria were identified as H. mustelaeby Gram's stain, morphology, strong oxidase, catalase, andurease positivity, sensitivity to nalidixic acid, and resistanceto cephalothin (10, 13).

Gastric pH. Gastric pH was monitored in four ferretsduring the two days of dosing and on days 7, 16, 34, 38, 49,56, 62, 94, and 162 post inoculation. The eight control ferretshad gastric pH measured on days 50, 57, and 99 of the study.

A pediatric pH probe (Synectics SYN-02 pH meter; Synec-tics Medical, Inc., Irving, Tex.) was used. Before the pHprobe was placed, ferrets were anesthetized with ketamineand acepromazine at the same doses used when administer-ing 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'tgiven.

Histopathologic examination. Antral and fundic biopsysamples for histopathologic examination were taken adja-cent to samples for bacterial culture and tissue urease.Tissue were fixed in neutral buffered 10% Formalin, proc-essed by standard methods, and embedded in paraffin;sections were cut 5 ,um thick and stained with hematoxylinand eosin (H&E) and by the Warthin-Starry method. Apathologist without foreknowledge of the source of thebiopsy specimens examined the sections for histologicalchanges and presence of H. mustelae.ELISA for H. mustelae antibody. Antigen was prepared

from whole-cell extracts by previously published methodsfor H. mustelae ELISA used in our laboratory (11, 12).Three isolates of H. mustelae (including ATCC 43772) fromgastric biopsy samples of ferrets were used. The ELISA wascarried out by the methods of Fox et al. (11, 12). The titer ofserum samples was expressed as the dilution of serum givinga reading equal to the mean plus 2 standard deviations (SD)of the negative control values.

Sera. Sera from H. mustelae-infected animals was col-lected at day 0 and 1, 2, 4, 8, 12, 14, and 24 weeks; controlsera from the eight SPF ferrets were taken at 8 and 12 weeks.

RESULTS

Colonization of the stomach with H. mustelae. The cultureand urease results from biopsy specimens of gastric antrumand fundus indicate that experimentally infected ferrets werecolonized with H. mustelae by 2 weeks p.i. Positive ureasetest results on antral and fundic tissue correlated well withthe positive H. mustelae cultures; the rapid urease reaction(within 2 h) in many biopsy samples indicated heavy coloni-zation (17) (Table 1). Warthin-Starry staining of stomachsamples also indicated colonization with H. mustelae in boththe antrum and fundus of all ferrets sampled on weeks 2through 24.

Figure 1 depicts H. mustelae in the stomach of an infectedferret. Short rod-shaped to slightly curved bacteria typical ofH. mustelae were present on the surface of the gastricepithelium within the mucus layer and within gastric pits.ELISA. At 2 weeks after H. mustelae inoculation, the four

INFECT. IMMUN.

on October 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

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. mustelaeantibody titers (<1:64) for the 12 ferrets (four experimentaland eight control) (Fig. 2). ELISA values on subsequentanalysis, during the 24 weeks of the study, indicated a steadyincrease in H. mustelae IgG antibody titers in the four H.mustelae-infected ferrets, whereas H. mustelae antibodytiters in the eight control ferrets remained <1:64.Measurement of gastric pH. Cimetidine was clearly effec-

tive in producing an apparent hypochlorhydria (as reflectedby elevated gastric pH) for the oral inoculation of H.mustelae on two successive days (Fig. 3). At weeks 1 and 2p.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; thepH measured 4 days later in all four ferrets was elevated (pH4.0 to 5.2). The raised gastric pH was transient (approxi-mately 2 weeks), as evidenced by a return to acidic pH in allferrets when measured 1, 2, and 4 weeks after the period ofapparent 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 controlferrets taken during weeks 8 and 12 of the study wereconspicuous by the uniform absence of any significant in-flammatory cell infiltration (Fig. 4). Minimal numbers oflymphocytes and occasional polymorphonuclear leukocyteswere scattered throughout the subglandular portions of boththe fundic and pyloric mucosa.

Biopsy specimens from the four H. mustelae-infectedferrets obtained at 1, 2, 4, 8, 12, and 24 weeks p.i. had focal,relatively mild inflammatory cell infiltrates in the pyloricmucosa of three of the four ferrets, whereas the fundicmucosa were similar to those from the controls except forthe presence of a focal superficial gastritis consisting oflymphocytes and occasional neutrophils. The inflammatory

in.0

I

1:100 00

1:1000/ N

1:100

< 1:64 1'

6-

P 4-

2-

Control 2 4 8 12 14 24

Week

FIG. 2. H. mustelae IgG titers in four experimentally infectedferrets. Each symbol represents an individual ferret: L, ferret 1; 0,ferret 2; ., ferret 3; *, ferret 4.

* 0 I I6 I I I I

1 7 1 6 34 38 49 56 62 94 162

Day Post Inoculation

FIG. 3. Gastric pH measurements in four ferrets experimentallyinfected with H. mustelae. Each symbol represents an individualferret. Values for SPF control ferrets are also shown (x). *, Ferretswere administered H. mustelae and cimetidine on days -1 and 0.

-1-i-0--- 2

-h- 3- - 4

VOL. 59, 1991

on October 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

1878 FOX ET AL.

44.^ Y bsM7Xt4FIG. 4. Typical example of the minimal numbers of leukocytes FIG. 5. Example of the magnitude of the predominantly lympho-

observed in samples of the gastric mucosa of ferrets free of H. cytic infiltration of the subglandular region of the gastric mucosamustelae. H&E. Magnification, x300. observed in samples from ferrets orally inoculated with H. muste-

lae. H&E. Magnification, x150.

cell infiltrates in the antrum were composed primarily oflymphocytes, with a few neutrophils and eosinophils inter-spersed. The foci of inflammation consisted of aggregates ofleukocytes primarily concentrated in the subglandular por-tion of the mucosa, with extension into the lamina propriatoward the luminal surface and through the muscularismucosa into the submucosa (Fig. 5 and 6). A few cysticglands were observed in selected pyloric biopsy samples, butthis was an inconsistent finding. From the biopsy samplesevaluated, there was no clearly defined evidence to suggesta significant increase in the severity of lesions during the6-month interval of the experiment.

DISCUSSION

Arguments supporting the contention that H. pylori is apathogen are based on several interrelated observations.Warren and Marshall's initial discovery that H. pylori wasassociated with gastritis was supported by further studiesshowing that in patients with H. pylori gastritis treated withantimicrobial agents, the gastritis resolved (30). The fulfill-ment of Koch's postulate with two human volunteers whoingested H. pylori and developed gastritis also favors therole of H. pylori as a gastric pathogen (23, 28). Morerecently, H. pylori has been associated with the develop-ment of duodenal ulcers (25, 33, 35). Efforts to study the

pathogenesis of H. pylori in animal models have beenhampered by the organism's host specificity. To date theonly reliable model for studying H. pylori infection has beenthe gnotobiotic pig (19). A recent report suggests that thegnotobiotic dog and SPF pig (raised under barrier condi-tions) may also be appropriate models for some H. pyloripathogenesis studies (7, 31). Preliminary evidence indicatesthat nonhuman primates may also be susceptible to H. pyloriinfection (2). Unfortunately, many of these species are alsocolonized with a nonculturable gastric spirillum, Gastro-spirillum hominis, which also occasionally infects humans(14, 27, 34). In addition, these models have disadvantages inregard to availability, housing, cost, and ease of administer-ing antimicrobial agents for eradication studies. Though therat and mouse are not colonized by H. pylori, we haveshown that H. felis colonizes and produces a gastritis in bothgnotobiotic rats and mice (14a, 20). These rodent models willafford the opportunity to study Helicobacter gastric infec-tion, but because of their size, the taking of repeated gastricbiopsy samples from individual animals is not feasible.The ferret offers several advantages for the study of

Helicobacter-induced gastritis. The ferret has a stomachwith anatomical and physiological characteristics similar tothose of humans; it is readily available commercially, is easyto maintain in a research setting, and has a tractable dispo-

INFECT. IMMUN.

on October 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

H. MUSTELAE INDUCES GASTRITIS IN FERRETS 1879

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 theferret stomach is naturally colonized by H. mustelae, whichis closely related phenotypically, by DNA homology andRNA sequencing data, and biochemically to H. pylori (9, 10,13). H. mustelae, like H. pylori, closely adheres to gastricmucosa and produces a gastritis similar to that noted inchildren and some adults infected with H. pylori (6, 12); alsowe have recently found that H. mustelae has urease subunitssimilar to those reported for H. pylori (6a, 11). Thesesimilarities are important because both adherence and ure-

ase production have been suggested to play a role in thepathogenesis of H. pylori gastroduodenal disease. Our orig-inal studies indicated that H. mustelae colonizes nearly100% of the ferrets we have sampled and that colonizationoccurs shortly after weaning. The infection is accompaniedby gastritis which persists in the stomach for years, probablyfor the life of the animal (9, 11). This prompted us to deriveand maintain a small nucleus of SPF ferrets without H.mustelae infection. Also, therapy studies indicate that erad-ication of H. mustelae is possible by using antimicrobialagent regimens similar to those used to eradicate H. pylori inhumans (29).The present study fulfills Koch's postulates and demon-

strates that H. mustelae is a gastric pathogen. The organism,inoculated into previously H. mustelae-negative ferrets,

readily colonized the gastric mucosa and produced an im-mune response and chronic gastritis, all features which arenoted in ferrets naturally colonized with H. mustelae (11,13). The stomach lesions, consisting of focal chronic antralgastritis induced by the experimental infection with H.mustelae, are similar to the gastritis observed in youngferrets that have been naturally colonized with the organismfor a limited time period (14b). The focal nature of thegastritis may explain the lack of significant inflammatoryresponse in one of the infected ferrets.Another intriguing finding in the current study was the

apparent transient hypochlorhydria observed approximately4 weeks after the infection. According to the urease tissueassay, this period coincided with heavy H. mustelae coloni-zation of the fundus. In the experimentally infected ferretsover time (>8 weeks), the organism, as in most naturallyinfected ferrets, colonizes the antrum in greater numbersthan the fundus (11). Of the two humans who voluntarilyingested H. pylori, one had confirmed elevated gastric pHlasting several weeks (28). The previous observation ofepidemic hypochlorhydria in volunteer patients undergoinggastric physiology studies is now thought to be related toacute infection with H. pylori (32). latrogenic acute H. pylorigastritis in another patient was accompanied by a similarepisode of achlorhydria noted during the second week ofinfection and lasting approximately 2 months (16).The mechanism which causes the transient gastric hy-

pochlorhyria observed in the H. mustelae-infected ferretsand in human cases of H. pylori gastritis is unknown.Marshall et al. recently demonstrated in vitro that H. pyloriis able to utilize physiological concentrations of urea suffi-cient to neutralize the hydrogen ions present in gastric juice(24). They suggested that the pH neutralization occurringduring H. pylori metabolism of urea via the action of ureasemay have been sufficient to explain the basal hypochlorhy-dria seen in the volunteer experiment described previously(24, 32). They theorized that the parietal cell failure may bedue to toxic effects of the ammonia production occurringduring 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 anondialyzable protein is responsible for the antisecretoryeffect on the parietal cell (4). We have recently demonstratedthat H. mustelae switches off both ferret and rabbit parietalcells in vitro (34a).The possibility of direct inhibitory action on parietal cells

is further supported by the observation of gastric spirilla inclose association with parietal cells in humans infected withH. pylori, in dogs, cats, and nonhuman primates infectedwith H. felis or Gastrospirillum hominis, and in rodentsexperimentally infected with H. felis (5, 18, 19a, 22, 34). Indogs, in which spiral organisms in parietal cells have beennoted frequently, the parietal cell caniculi were sometimesdilated, and occasionally parietal cells were vacuolated anddegenerated (18). Further studies are needed to prove ordisprove the proposed theories regarding the pathogenesis ofHelicobacter-associated hypochlorhydria.

In conclusion, H. mustelae infection in the ferret hasmany of the same features observed in H. pylori-associatedgastritis in humans. Our observations concerning naturallyoccurring H. mustelae gastritis in ferrets have been repro-duced by experimental infection with H. mustelae in SPFferrets. The ferrets developed an infection which resulted ina persistent, chronic gastritis and significant immune re-sponse. The additional feature of elevated gastric pH in H.mustelae-infected ferrets further supports the use of this

VOL. 59, 1991

on October 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

1880 FOX ET AL.

model to study the natural progression and pathogenesis ofHelicobacter-induced gastritis in mammalian hosts.

ACKNOWLEDGMENTS

This research was supported in part by Public Health Servicegrants R01-A125631 from the National Institute of Allergy andInfectious Diseases, P01-CA-26731 from the National Cancer Insti-tute, and RR01046 and T32-RR07036 from the Center for ResearchResources.

REFERENCES1. Barthel, J. S., T. V. Westblom, A. D. Havey, F. Gonzalez, and

E. D. Everett. 1988. Gastritis and Campylobacter pylori inhealthy asymptomatic volunteers. Arch. Intern. Med. 148:1149-1151.

2. Baskerville, A., and D. B. Newell. 1988. Naturally occurringchronic gastritis and C. pylori infection in the rhesus monkey: apotential model for gastritis in man. Gut 29:465-472.

3. Cantorna, M. T., and E. Balish. 1990. Inability of human clinicalstrains of Helicobacter pylori to colonize the alimentary tract ofgerm-free rodents. Can. J. Microbiol. 36:237-241.

4. Cave, D. R., and M. Vargas. 1989. Effect of a Campylobacterpylori protein on acid secretion by parietal cells. Lancet ii:187-188.

5. Chen, X. G., P. Correa, J. Offerhaus, E. Rodriguez, F. Janney,E. Hoffmann, J. Fox, F. Hunter, and S. Diavolitsis. 1986.Ultrastructure of the gastric mucosa harboring Campylobacter-like organisms. Am. J. Clin. Pathol. 86:575-582.

6. Czinn, S. J., B. B. Dahms, G. H. Jacobs, B. Kaplan, and F. C.Rothstein. 1986. Campylobacter-like organisms in associationwith symptomatic gastritis in children. J. Pediatr. 109:80-83.

6a.Dunn, B. E., C. C. Sung, N. S. Taylor, and J. G. Fox. Submittedfor publication.

7. Engstrand, L., S. Gustavsson, A. Jorgensen, A. Schwan, and A.Scheynius. 1990. Inoculation of barrier-born pigs with Helico-bacter pylori: a useful animal model for gastritis type B. Infect.Immun. 58:1763-1768.

8. Fox, J. G. 1988. Biology and diseases of the ferret. Lea andFebiger, Philadelphia.

9. Fox, J. G., E. B. Cabot, N. S. Taylor, and R. Laraway. 1988.Gastric colonization of Campylobacter pylori subsp. mustelaein ferrets. Infect. Immun. 56:2994-2996.

10. Fox, J. G., T. Chilvers, C. S. Goodwin, N. S. Taylor, P.Edmonds, L. I. Sly, and D. J. Brenner. 1989. Campylobactermustelae, a new species resulting from the elevation of Cam-pylobacterpylori subsp. mustelae to species status. Int. J. Syst.Bacteriol. 39:301-303.

11. Fox, J. G., P. Correa, N. S. Taylor, A. Lee, G. Otto, J. C.Murphy, and R. Rose. 1990. Helicobacter mustelae-associatedgastritis in ferrets: an animal model of Helicobacter pylorigastritis in humans. Gastroenterology 99:352-361.

12. Fox, J. G., P. Correa, N. S. Taylor, D. Zavala, E. Fontham, F.Janney, E. Rodriguez, F. Hunter, and S. Diavolitsis. 1989.Campylobacter pylori associated gastritis and immune responsein a population at increased risk of gastric carcinoma. Am. J.Gastroenterol. 89:775-781.

13. Fox, J. G., B. M. Edrise, E. B. Cabot, C. Beaucage, J. C.Murphy, and K. S. Prostak. 1986. Campylobacter-like organ-isms isolated from gastric mucosa of ferrets. Am. J. Vet. Res.47:236-239.

14. Fox, J. G., and A. Lee. 1989. Gastric campylobacter-like organ-isms: their role in gastric disease in laboratory animals. Lab.Anim. Sci. 39:543-553.

14a.Fox, J. G., A. Lee, G. Otto, N. S. Taylor, and J. C. Murphy.1991. Helicobacter felis gastritis in gnotobiotic rats: an animalmodel of Helicobacter pylori gastritis. Infect. Immun. 59:785-791.

14b.Fox, J. G., G. Otto, J. C. Murphy, N. S. Taylor, and A. Lee.Rev. Infect. Dis., in press.

15. Goodwin, C. S., J. A. Armstrong, T. Chilvers, M. Peters, M. D.Collins, L. Sly, W. McConnell, and W. E. S. Harper. 1989.

Transfer of Campylobacter pylori to Helicobacter gen. nov. asHelicobacter pylori comb. nov. and Helicobacter mustelaecomb. nov., respectively. Int. J. Syst. Bacteriol. 39:397-405.

16. Graham, D. Y., M. D. Lesley, C. Alpert, J. L. Smith, and H. H.Yoshimura. 1988. latrogenic Campylobacter pylori infection is acause of epidemic achlorhydria. Am. J. Gastroenterol. 83:974-980.

17. Hazell, S. L., T. J. Brody, A. Gal, and A. Lee. 1987. Campylo-bacter pyloridis gastritis. I. Detection of urease as a marker ofbacterial colonization and gastritis. Am. J. Gastroenterol. 82:292-296.

18. Henry, G. A., P. H. Long, J. L. Burns, and D. L. Charbonneau.1987. Gastric spirillosis in beagles. Am. J. Vet. Res. 48:831-836.

19. Krakowka, S., D. R. Morgan, W. G. Kraft, and R. D. Leunk.1987. Establishment of gastric Campylobacter pylori infection inthe neonatal gnotobiotic piglet. Infect. Immun. 55:2789-2796.

19a.Lee, A., and J. G. Fox. Unpublished data.20. Lee, A., J. G. Fox, G. Otto, and J. Murphy. 1990. A small animal

model of human Helicobacter pylori active chronic gastritis.Gastroenterology 99:1315-1323.

21. Lee, A., S. L. Hazell, J. O'Rourke, and S. Kouprach. 1988.Isolation of a spiral-shaped bacterium from the cat stomach.Infect. Immun. 56:2843-2850.

22. Lockard, V. G., and R. K. Boler. 1970. Ultrastructure of aspiraled microorganism in the gastric mucosa of dogs. Am. J.Vet. Res. 31:1453-1462.

23. Marshall, B. J., J. A. Armstrong, D. B. McGechie, and R. J.Glancy. 1985. Attempt to fulfill Koch's postulates for pyloriccampylobacter. Med. J. Aust. 142:436-439.

24. Marshall, B. J., L. J. Barrett, C. Prakash, R. W. McCallum, andR. L. Guerrant. 1990. Urea protects Helicobacter (Campylo-bacter) pylori from the bactericidal effect of acid. Gastroenter-ology 99:697-702.

25. Marshall, B. J., C. S. Goodwin, J. R. Warren, R. Murray, E. D.Blincow, S. J. Blackbourn, M. Phil!ips, T. E. Waters, and C. R.Sanderson. 1988. Prospective double-blind trail of duodenalulcer relapse after eradication of Campylobacter pylori. Lancetii:1437-1442.

26. Marshall, B. J., and J. R. Warren. 1984. Unidentified curvedbacilli in the stomach of patients with gastritis and pepticulceration. Lancet i:1311-1315.

27. McNulty, C. A., J. C. Dent, A. Curry, J. S. Uff, G. A. Ford,M. W. Gear, and S. P. Wilkinson. 1989. New spiral bacterium ingastric mucosa. J. Clin. Pathol. 42:585-591.

28. Morris, A., and G. Nicholson. 1987. Ingestion of Campylobacterpyloridis causes gastritis and raised fasting gastic pH. Am. J.Gastroenterol. 82:192-199.

29. Otto, G., J. G. Fox, P.-Y. Wu, and N. S. Taylor. 1990.Eradication of Helicobacter mustelae from the ferret stomach:an animal model of Helicobacter (Campylobacter) pylori che-motherapy. Antimicrob. Agents Chemother. 34:1232-1236.

30. Parsonnet, J. 1989. The epidemiology of C. pylori, p. 51-60. InM. J. Blaser (ed.), Campylobacter pylori in gastritis and pepticulcer disease. Igaku-Shoin, New York.

31. Radin, J. M., K. A. Eaton, S. Krakowka, D. R. Morgan, A. Lee,G. Otto, and J. G. Fox. 1990. Helicobacter pylori in gnotobioticbeagle dogs. Infect. Immun. 58:2006-2612.

32. Ramsey, E. J., K. V. Carey, W. L. Peterson, J. J. Jackson, F. K.Murphy, N. W. Read, K. B. Taylor, J. S. Trier, and J. S.Fordtran. 1979. Epidemic gastritis with hypochlorhydria. Gas-troenterology 76:1449-1457.

33. Rauws, E. A. J., and G. N. J. Tytgat. 1990. Cure of duodenalulcer associated with eradication of Helicobacter pylori. Lancet335:1233-1235.

34. Sato, T., and A. Takeuchi. 1982. Infection by spirilla in thestomach of the rhesus monkey. Vet. Pathol. 19:17-25.

34a.Vargas, M., A. Lee, J. G. Fox, and D. Cave. Submitted forpublication.

35. Wyatt, J. I. 1989. Relationship of C. pylori to duodenal ulcerdisease, p. 99-114. In M. J. Blaser (ed.), Campylobacter pyloriin gastritis and peptic ulcer disease. Igaku-Shoin, New York.

INFECT. IMMUN.

on October 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from