JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1990, p. 2285-22900095-1137/90/102285-06$02.00/0Copyright © 1990, American Society for Microbiology

Vol. 28, No. 10

Evaluation of Two Monkey Species (Macaca mulatta and Macacafascicularis) as Possible Models for Human

Helicobacter pylori DiseaseARTHUR R. EULER,t* GARY E. ZURENKO, JAMES B. MOE, ROGER G. ULRICH, AND YOSHI YAGI

Upjohn Laboratories, The Upjohn Company, Kalamazoo, Michigan 49001

Received 18 December 1989/Accepted 28 June 1990

Endoscopic, histologie, and microbiologie evaluations of 21 cynomolgus and 34 rhesus monkeys for naturallyoccurring Helicobacter pylori infection were done. H. pylori was never isolated from any cynomolgus monkey,but was found in 12 rhesus monkeys. A general correlation existed between a positive culture and a gastricinflammatory response. Inoculation challenges were then undertaken. Four cynomolgus and five rhesusmonkeys received two different H. pylori strains isolated from humans. Five rhesus monkeys received an isolateobtained from rhesus monkeys. Evaluation of the cynomolgus monkeys 7 and 14 days later revealed no H.pylori. Endoscopies of the rhesus monkeys were done 7, 14, 21, 28, and 56 days later. One rhesus monkey,which received the isolate from humans, became H. pylori positive at day 21 and remained positive through day56. Restriction enzyme analysis of genomic DNA at day 56 revealed that the isolate was not identical to thechallenge strain isolated from humans. All five rhesus monkeys that received the strain isolated from rhesusmonkeys became H. pylori positive by day 14 and remained positive through day 56. Antral inflammationdeveloped in all monkeys. Restriction enzyme analysis of genomic DNA on day 56 confirmed that four of fiveisolates were identical to the challenge strain isolated from rhesus monkeys. DNA hybridization documentedhomology between the challenge strains isolated from humans and rhesus monkeys plus those isolated at day56. In this study, we showed that the rhesus monkey, if given a strain ofH. pylori isolated from rhesus monkeys,develops a gastric infection with accompanying histological changes, making this model suitable for furtherdevelopment.

Since the initial report of Warren and Marshall (23) of"unidentified curved bacilli" on the gastric epithelium ofhumans, multiple reports have documented the epidemiol-ogy of human disease associated with Helicobacter pylori(Campylobacter pylori) (5, 10, 18). In vitro susceptibilitytests of multiple strains of H. pylori have shown that it issusceptibile to many antibiotics (8, 12). Although the organ-ism is very susceptible to a variety of antibiotics, clinicaltrials in which monotherapy was used with many of theseantibiotics have failed to show good eradication rates (7, 9;R. Gilman, R. Leon-Barua, A. Ramirez-Ramos, D. Morgan,S. Recavarron, W. Spira, P. Watanabe, W. Kraft, and A.Pearson, Gastroenterology 92:1405, 1987). These clinicalresults and the desire to learn more about the pathogenicityand pathophysiology associated with H. pylori infectionsmake the establishment of an animal model a critical need. Anumber of investigators have reported the isolation of H.pylori during evaluations of different monkey species (1, 3,21). We therefore undertook studies in a large number ofMacaca fascicularis (cynomolgus) and Macaca mulatta(rhesus) monkeys to determine whether either would serveas a suitable model ofH. pylori disease in humans. We chosethese two species since the cynomolgus monkey is muchmore available and would enable more investigators to usethe model, if it was established, while the rhesus monkey ismost closely related to humans, although its availability islimited. Here we report the results of extensive investiga-tions in which we used endoscopy, necropsy, light micros-copy, scanning and transmission electron microscopy

* Corresponding author.t Present address: Glaxo, Inc., Five Moore Drive, Research

Triangle Park, NC 27709.

(TEM), and microbiological culture techniques. These meth-ods were used both during the initial screening of these twospecies to determine the incidence of naturally occurring H.pylori infections as well as during subsequent challengeexperiments.

MATERIALS AND METHODSAnimals. We initially evaluated 21 adult cynomolgus and

34 adult rhesus monkeys for the presence of naturallyoccurring H. pylori infections. The endoscopic procedureconsisted of esophagogastroduodenoscopy, with biopsyspecimens being obtained from the gastric fundus and an-trum. Prior to the procedure, the monkeys were fasted forapproximately 18 h and then sedated with ketamine hydro-chloride (50 to 100 mg intramuscularly; Vetalar; Parke,Davis & Co., Morris Plains, N.J.). These procedures wereperformed with an Olympus GIF P2 endoscope. Endoscopywas performed in 25 cynomolgus monkeys, and 200 biopsyspecimens were obtained. Four animals were each examinedtwice by endoscopy. Four fundic and antral biopsy speci-mens were obtained for microbiological culture, rapid tissueurease testing, and light and electron microscopy. Necropywas performed in 19 cynomolgus monkeys, and 304 biopsyspecimens were obtained. Four specimens were obtainedfrom each monkey. One specimen each was obtained fromthe esophagus, fundus, antrum, and duodenum. The testslisted above for endoscopy in cynomolgus monkeys wereperformed. Endoscopy was performed in 34 rhesus mon-keys, and 272 biopsy specimens were obtained as describedabove. Necropsy was performed in 1 rhesus monkey, and 16biopsy specimens were obtained. Endoscopy was not per-formed in this rhesus monkey.

Histology. Necropsy and biopsy specimens from the

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esophagus, stomach, and duodenum were fixed in 10%neutral buffered Formalin, trimmed, processed, and embed-ded in paraffin. Replicate 4- to 6-,um sections of eachspecimen were mounted on glass slides; one section wasstained with hematoxylin-eosin-phloxine for morphology,and the other was stained with Warthin-Starry silver stainfor microorganisms. Slides were examined for histopatho-logic evidence of epithelial changes, inflammation, and thepresence of microorganisms.

Microbiology. One biopsy specimen was transferred fromthe transport vial containing normal saline to a sterile petridish, where it was minced with sterile surgical scissors. Thesecond biopsy specimen was transferred to 0.5 ml of ureasereagent (Rapid Urea; Remel Corp., Lenexa, Kans.) andincubated at 35°C for 1 h, at which time the test result wasread. The minced tissue was transferred to the surface of anagar medium plate which contained Skirrow medium base(Difco Laboratories, Detroit, Mich.) supplemented with 7%difibrinated horse blood and to a microscope slide for lightmicroscopic examination following Gram staining. Carbol-fuchsin was substituted for safranin as the counterstain inthe Gram stain procedure. Culture plates were incubated for7 days at 35°C in a microaerobic environment (CampyPakplus palladium catalyst in a anaerobicjar; BBL MicrobiologySystems, Cockeysville, Md.) and examined at 4 and 7 days;representative colony types were screened for urease pro-duction (Rapid Urea), and if they were positive, they weresubcultured for biochemical identification. Isolates werecharacterized by their macroscopic and microscopic mor-phologies; the results of urease, catalase, and oxidase tests;as well as their biochemical profiles by using the Rapid NHsystem (Innovative Diagnostics, Inc., Atlanta, Ga.) (22).Animal challenge studies. (i) Cynomolgus monkeys. After

the initial endoscopic studies were completed, an attempt toinfect four cynomolgus monkeys orally with a fresh H. pyloristrain from a human was undertaken. H. pylori 30785 wasisolated from an antral biopsy specimen from a human onSkirrow medium base (supplemented with 7% defibrinatedhorse blood). The culture was passaged once on Columbiaagar base (supplemented with 7% defibrinated horse blood)for purification and identification, and cells from this passagewere transferred directly to broth medium from a prepara-tion of the animal inoculum. Briefly, several colonies wereharvested with a sterile cotton swab and used to inoculate 25ml of supplemented brucella broth consisting of brucellabroth (Difco) plus 1% fetal bovine serum (GIBCO Labora-tories, Grand Island, N.Y.) and 1% IsoVitaleX (BBL) con-tained in a 125-ml screw-cap Erlenmeyer flask with a loose-fitting cap. The flasks were taped to the bottom of a largeanaerobe jar (BBL) and incubated under microaerobic con-ditions (as described above) for 72 h on a rocker platform.The cells were concentrated 10-fold by centrifugation andsuspended in alkaline peptone water. The number of CFUper milliliter was determined by preparing serial 10-folddilutions of the concentrated cell suspension and platingaliquots onto duplicate agar plates (Columbia agar basesupplemented with 7% defibrinated horse blood). Followingincubation, colonies were counted and the final cell concen-tration was determined. The concentration of the H. pylori30785 cell suspension used to inoculate the cynomolgusmonkeys was 109 CFU/ml.The animals were fasted for 24 h and then given a 5-ml

volume of the inoculum described above. The inoculum wasgiven through a nasogastric tube, and then 5 ml of distilledwater was administered by the same route. The animals werefed immediately after receiving the inoculum. At 7 days

postchallenge, endoscopy was performed, during which fun-dic and antral biopsy specimens were obtained for H. pyloriculture, rapid tissue urease testing, and light and electronmicroscopy.

(ii) Rhesus monkeys. The 23 rhesus monkeys identified asbeing H. pylori negative during our initial screening werereendoscoped 7 days prior to the challenge experiment toconfirm they were truly negative for H. pylori. Based on theresults of our initial screening endoscopic examinations,which showed that evidence of H. pylori infection in theantrum was documented whenever the organism was foundin the gastric fundus, biopsy specimens were taken onlyfrom the gastric antrum during these and subsequent biop-sies. Three antral biopsy specimens were taken from eachanimal and processed as described above. Two biopsyspecimens were used for bacterial culture, and the thirdbiopsy specimen was processed for light microscopy. Biopsyspecimens were obtained for electron micropsy duringesophagogastroduodenoscopies from areas of endoscopi-cally evident mucosal damage in the stomach, duodenum, orboth. After the results of the repeat endoscopic studies wereavailable, 10 rhesus monkeys which were negative for H.pylori in both examinations were chosen for the bacterialchallenge experiment. The animals were divided into twogroups of five each.One group was orally inoculated with H. pylori UC12228

(isolate from a human), while the other group was inoculatedwith H. pylori UC12476 (isolate from a rhesus monkey). H.pylori UC12228 was provided by T. Ulf Westblom (MarshallUniversity, Huntington, W.V.), having originally been iso-lated from a symptomatic patient. This culture has under-gone a number of in vitro passages and was maintainedfrozen in the vapor phase of a liquid nitrogen freezer prior touse in our study. H. pylori UC12476 was isolated from arhesus monkey during our initial screening. This isolate wasalso maintained frozen until use, having undergone only fourin vitro passages. Cell suspensions were prepared for each ofthese cultures by the same method described above for H.pylori 30785. The final cell concentrations for H. pyloriUC12228 and H. pylori UC12476 were 2 x 108 and 2 x 106CFU/ml, respectively.The animals were fasted for 24 h before they received a

3-ml volume of inoculum via a nasogastric tube, and then 5ml of distilled water was administered by the same route.The monkeys were fed immediately after the inoculum wasgiven. Endoscopies were performed at 7, 14, 21, 28, and 56days postinoculation. During each of these endoscopies,three antral biopsy specimens were obtained and processedas described above. Antibody studies were not performed.TEM. Samples were prepared for TEM by suspending

them in 0.1 M cacodylate buffer (pH, >2.0) containing 3.0%glutaraldehyde and 5% sucrose. After 1 h, samples werepelleted and then rinsed twice with 0.1 M cacodylate buffercontaining 5% sucrose. Samples were applied to 400-meshgrids containing a nitrocellulose support film by floating thegrid on a drop ofconcentrated suspension. Excess liquid wasremoved from the grids, and samples were allowed to airdry. Negative stain (1% phosphotungstic acid) was thensimilarly applied. Samples were examined by TEM in aJEOL 1200 EX instrument at 80 keV.

For negative staining, samples of cultured H. pylori weresuspended in phosphate-buffered saline and fixed by theaddition of 70% glutaraldehyde to a final concentration of3%. Following fixation, samples were treated as describedabove.DNA homology. To obtain total DNA from each isolate,

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TABLE 1. Endoscopic observations in 34 rhesus monkeysevaluated dunng initial screenings

Endoscopic observation No. of monkeys

Normal ....................................... 19Antral erythema (mild to moderate) ...................... 8Halo lesions .................. ..................... 3Antral erythema and hemorrhages (n = 5) ........ ..... 1Antral gastritis ..................... .................. 2Antral erosions ....................... ................ 1

a Small papules with pale center surrounded by erythema.

we used a previously described method (4) with the follow-ing modification. Cells were harvested from a 90-mm-diam-eter plate (Columbia agar medium) with a cotton swab, anda cell suspension was made with 1.0 ml of TES (0.03 MTris-0.005 M disodium EDTA-0.05 M NaCl [pH 8.0]) buffer.RNase A (0.2 ml of a 5-mg/ml solution; Sigma Chemical Co.,St. Louis, Mo.) was added prior to the addition of pronase(0.2 ml of a 5-mg/ml solution in TES buffer predigested at37°C for 30 min) (Calbiochem-Behring, La Jolla, Calif.).Lysis was achieved by the addition of 0.2 ml of 10% sodiumdodecyl sulfate. Standard protocols were used for DNA-DNA hybridization, as well as analysis of genomic DNA byusing the restriction endonuclease BamHI (15). By usingrandom priming reagents from Boeringer Mannheim Bio-chemicals (Indianapolis, Ind.), 32P-labeled probes were pre-pared according to the instructions of the manufacturer.

RESULTSEndoscopy and necropsy. All endoscopic and gross nec-

ropsy examinations of cynomolgus monkeys revealed noabnormalities except in one animal, which had a small antralpolyp (diameter, 0.5 cm). The necropsy examination of therhesus monkey revealed gross abnormalities. The observa-tions made during the initial screening endoscopic examina-tions in the rhesus monkey are given in Table 1. Theendoscopic observations at 7, 14, 21, 28, and 56 days afterthe inoculation attempt with the human strain in the rhesusmonkey are given in Table 2. The endoscopic results atidentical time intervals after the inoculation attempt with thestrain from a rhesus monkey are given in Table 3. Theendoscopic findings varied over time within individual ani-mals. We believe this was due to the severity of the infectionand the animal's response to such infections.

TABLE 3. Culture results and gastric inflammation scores forfive H. pylori-negative rhesus monkeys infected with a

H. pylori strain of rhesus origin (UC12476)

Culture result/inflammation score at the followingMonkey postinfection interval (days)a:

0 7 14 21 28 56

1 0/0 0/0 1/2 0/1 1/2 1/02 0/0 0/1 1/1 1/0 1/3 1/33 0/0 1/0 0/0 1/0 1/1 1/04 0/0 1/0 0/0 1/0 0/3 1/05 0/2 1/2 1/3 1/2 1/3 1/2

a Results are those for two biopsy specimens; a positive culture result foreither biopsy specimen was defined as a culture-positive animal. Inflammationscores: 0, no inflammation; 1, mild inflammation; 2, moderate inflammation; 3,severe inflammation.

Histology. There were no histopathologic abnormalities inthe esophagi or duodenums of the cynomolgus monkeysfrom which specimens were obtained. There was also noevidence of epithelial alteration in any cynomolgus monkey,except for one monkey which had a hyperplastic gastricpolyp containing sections of the nematode parasite Nochtianochtia.There was a noticeable mixed mononuclear ceil inflamma-

tory response in the stomachs of 14 of the 35 rhesusmonkeys. This response consisted principally of a lymphoidinfiltration of the superficial layers of the lamina propria ofthe fundic and antral regions (Fig. 1). Additionally, muci-nous hyperplasia of the surface epithelium occurred in thestomachs of eight rhesus monkeys, while focal squamousmetaplasia was found in one rhesus monkey (Fig. 2). It wasnot possible in any case to confidently identify organismscompatible with H. pylori in paraffin-embedded tissue sec-tions.

Electron microscopy. TEM revealed that the isolates of H.pylori from a human and a rhesus monkey given in theinoculum and the isolates obtained during the subsequentendoscopies in the group of five monkeys that received therhesus strain were essentially identical in negative stainpreparations (Fig. 3). The human isolate was 1.3 to 2.7 ,um inlength (mean, 2.1 + 0.4 k.m) and 0.4 ,um in diameter. Therhesus isolate was 1.7 to 2.7 ,um in length (mean, 2.2 + 0.4,um) and 0.4 ,um in diameter (Fig. 3). Both strains exhibitedfour flagella at a single pole.

Microbiology. Campylobacter-like organisms subse-

TABLE 2. Endoscopic observations after inoculation attempt in 10 rhesus monkeys

Endoscopic observation on the following day after inoculationsMonkey

7 14 21 28 56

1 WNL EA PWP (5) and antral PWP (3) and antral EA and antralhemorrhage petechiae petechiae

2 WNL Antral PWP (7) PWP (10) PWP (12) and EA PWP (12)3 WNL PWP (10) WNL PWP (10) and EA PWP (5)4 EA EA and PWP (7) WNL PWP (10) PWP (13)5 EA EA and PWP (8) WNL PWP (16) PWP (11)6 WNL WNL WNL WNL WNL7 WNL WNL EA Friability and antral ulcer Antral gastritis and

(diam, 1.0 cm) ulcer healed8 WNL WNL EA and antral EA and antral hemorrhages Antral gastritis

hemorrhages (5) (6) and PWP (4)9 PWP (5) PWP (8) PWP (10) PWP (18) PWP (7)10 PWP (10) PWP (10) PWP (12) PWP (19) PWP (15)

a Five monkeys (1 to 5) received human H. pylori strain, and five monkeys (6 to 10) received rhesus monkey H. pylori strain.b Abbreviations: WNL, normal; EA, erythematous antrum; PWP, pale white papules; numbers in parentheses are number of lesions.

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# ,' .-Yl la

FIG. 1. Light micrograph of a mononuclear inflammatory infli-

trate in the superficial region of the gastric mucosa of a rhesus

monkey infected with H. pylori; hematoxylin-eosin-phloxine stain

was used. Bar, 40 p.m.

quently identified as H. pylori were isolated from of 21

cynomoigus and 12 of 35 rhesus monkeys. These cultures

grew as nonpigmented colonies on both Skirrow-based and

Coiumbia-based agars under microaerobic incubation condi-

tions; light microscopic examination showed that the organ-

isms were curved, gram-negative rods. The organisms were

strongly urease positive resultt within 5 min) and catalase

and oxidase positive.

F

FIG. 2. Light micrograph of focal squamous metaplasia of the

gastric surface epithelium in a rhesus monkey infected with H.

pylori; hematoxylin-eosin-phloxine stain was used. Bar, 40 ~tm.

Correlation of the isolation of H. pylori with histologicalevidence of gastric inflammation was not possible in thecynomolgus monkeys, as there were no culture-positive orinflammation-positive animals in the group surveyed. How-ever, in the rhesus monkeys, a strong correlation did exist.Histological evidence of inflammation was seen in 10 of 12(83%) culture-positive animals, but only in 4 of 23 (17%)culture-negative animals. Endoscopie observations were notgenerally predictive of either culture or histopathoiogy re-suits. The rapid tissue urease test results (data not shown)were not predictive of H. pylori infection in monkeys be-cause of a high percentage of false-positive results.

Experimental infections. In the inoculation experiment inthe rhesus monkeys, five H. pylori-negative rhesus monkeyswere inoculated with a strain of H. pylori from either ahuman (H. pylori UC12228) or a rhesus monkey (H. pyloriUC12476). Four of five animals inoculated with the strainfrom a human remained H. pylori negative through 8 weeks.One animal became H. pylori-positive at 2 weeks; however,virtually no gastric inflammation developed despite the pres-ence of the organism. We observed no noticeable endo-scopic changes. DNA-DNA hybridization indicated that theisolate was highly homologus (80%) to the strain from ahuman that was used for inoculation; however, restrictionendonuclease (BamHI) analysis revealed that they were notidentical, and the isolate may have been an undetectedendogenous strain.

In contrast to these findings, five of five monkeys inocu-lated with the isolate from the rhesus monkey became H.pylori positive by 14 days postinfection, and ail five animalsdemonstrated inflammation by 28 days postinfection. Theorganism persisted during the entire 56-day interval (Table3). The postinoculation isolates from these animals exhibiteda high degree of homology (70 to 100%) to the challengeisolate. Subsequent analysis with a restriction enzyme(BamHI) indicated that four of the isolates were identical tothe challenge isolate, while one was slightly different. Acurious finding (data not shown) was that H. pylori couldoften be cultured from one antrai biopsy specimen, whiie theother specimen was negative. These results indicate that theinfection may not be uniform throughout the antrum andsuggest that multiple biopsy specimens should be used in ailendoscopic studies involving this monkey species. This mayexplain the occasional result of culture negativity in inflam-mation-positive animals (Table 3). Colonization of the antralmucosa by H. pylori was generaiiy accompanied by histo-logical evidence of inflammation. At 28 and 56 days postin-fection, there was striking histopathologicai evidence ofinflammation. One animal progressed to the development ofan antral ulcer at 4 weeks, which subsequently healedspontaneously by week 8. The isolate from this animal,however, was not identical to the challenge strain, as deter-mined by restriction enzyme analysis.

DISCUSSION

H. pylori has been documented as one of the importantfactors associated with type B gastritis and gastric andduodenal ulcer formation in humans (5, 11, 18). Although theexact pathophysiologicai processes underlying its pathoge-nicity are unknown, ingestion of the organism by volunteershas led to the development of gastric pathology and symp-toms (16, 19). An outbreak of H. pylori disease associatedwith sustained hypochlorhydria among volunteers undergo-ing gastric acid analysis has aiso been reported previously(20). Since these studies established the pathogenicity of the

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FIG. 3. Transmission electron micrograph of H. pylori (isolate from a rhesus monkey) prepared by negative staining. Four flagella areobserved at one pole. Bar, 1.0 ,um.

microorganism, many clinical trials in which antibioticmonotherapy based on antibiotic susceptibility testing hasbeen used have been undertaken without success (7, 9;Gilman et al., Gastroenterology). To date, the best therapyremains that involving three antibacterial agents (a bismuthcompound, an antibiotic such as amoxicillin, and a metron-idazole-like compound) (17). Although effective, this tripleantibacterial agent therapy is cumbersome and a problem forsome patients to take for the prescribed period because ofthe multiple daily dosings and side effects. Therapy with asingle antibiotic would have obvious advantages. Since invitro testing has not been shown to have a good clinicalcorrelation, the need exists for an animal model. Our eval-uations documented the fact that the monkey species chosenfor such model development is extremely important. Natu-rally occurring H. pylori infections were not present in ourcynomolgus monkeys, nor were we able to infect thismonkey species with an H. pylori strain isolated from ahuman. In contrast, we observed the incidence of naturallyoccurring H. pylori infections in the rhesus monkeys to beapproximately 34% (12 of 35 monkeys), with 10 of the 12monkeys having histological evidence of gastritis. Therhesus monkey was also susceptible to experimental infec-tions with a strain of H. pylori isolated from a rhesusmonkey.A number of previous studies have reported the isolation

of H. pylori in the rhesus monkey (1, 3, 21). Two of thesestudies used necropsy specimens; Baskerville and Newell (1)examined 11 animals, while Reed and Berridge (21) evalu-ated 7 animals. In the latter report, gross abnormalities(atrophy) were found in two monkeys. The remaining ani-mals' stomachs were normal on gross inspection. In thestudy of Baskerville and Newell (1), no gross findings except

erythema were reported, and its incidence was not noted. Inour study, the majority of rhesus monkeys infected with H.pylori had endoscopic findings compatible with acute type Bantral gastritis or halo-type lesions in the same gastricsegment. None had evident atrophic gastritis on endoscopicexamination. Histologically, none of our monkeys had atro-phic gastritis. The gastric inflammatory infiltrates reportedby the researchers mentioned above consisted of lympho-cytes, plasma cells, and histiocytes primarily in the antrum.Our findings were similar regarding these chronic inflamma-tory cells and their locations, but we also found that poly-morphonuclear leukocytes were frequently present. H. py-lori colonization occurred in 4 of 7 monkeys in the study ofReed and Berridge (21), while it occurred in 6 of 11 monkeysin the study of Baskerville and Newell (1). In the latterstudy, H. pylori was also not found in five cynomolgusmonkeys. The reason for the differences in histologicalfindings between those studies and this one is not evident,except for the obvious differences in technique in obtainingthe gastric specimens and the number of rhesus monkeysexamined.

All of our rhesus monkeys either had been born in ourcolony or had resided there for over 5 years. We tested theH. pylori strains isolated from our monkeys to determinewhether they were of human origin. The data suggest, but donot definitely confirm, that they were of human origin.Morphological, biochemical, and DNA homology testingdemonstrated that the H. pylori isolates from our monkeyswere identical to the H. pylori isolates from humans. TheDNA-DNA hybridization technique used for identification ofthe organism and the restriction endonuclease analysis usedfor examining the genomic arrangements showed that all theisolates were highly homologous, but not ail of them were

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identical. As has been noted by others (13, 14), such varia-tions in genomic structure are not uncommon. Previousreports (3, 6) have documented that the strains isolated fromthe rhesus monkey and Macaca nemestrina were of humanorigin. In those studies and this one, the presence of H.pylori was associated with an inflammatory cell response inthe gastric mucosa. In contrast, a histopathological study ofthe gastroduodenal mucosae in 100 rhesus monkeys exam-ined within 5 days of capture in the wild found inflammatoryor degenerative changes in only five animals (2). Althoughthat study (2) was published before the initial report ofWarren and Marshall (23) was, the low incidence of histo-pathology is striking compared with those in studies in whichH. pylori has been found (1, 3, 6, 21). The unique aspect ofthis histopathological study is that all animals underwentnecropsy within 5 days of being captured in the wild. Thesestudies would seem to indicate that, in rhesus monkeys, H.pylori is an acquired disease that probably results fromhuman contact.

Results of this study of cynomolgus and rhesus monkeys,which may be the largest to date, allowed us to make anumber of conclusions. Both monkey species can be easilyand repeatedly examined by endoscopy by well-trainedinvestigators without mortality or evident morbidity. H.pylori was found as a naturally occurring infection only inthe rhesus monkey and appears to be a gastric pathogen forthis species. Neither species was susceptible to experimentalinfection by human H. pylori strains in this study. However,the rhesus monkey was extremely susceptible to infection bya H. pylori strain of rhesus monkey origin, implying somelevel of strain adaptation in the animals; the cynomolgusmonkey was not challenged with the isolate from a rhesusmonkey in this study. While the cynomolgus monkey ap-pears to be less susceptible to H. pylori infection than therhesus monkey is, additional studies are needed to confirmthis observation. The rhesus monkey model described herewill enable us not only to evaluate antibiotic efficacies butalso to study the pathogenicity of H. pylori.

ACKNOWLEDGMENTS

We thank Ronda Schaadt and Betty Hannon Yagi for microbio-logical assistance, Phillip Roehm for invaluable help during theendoscopic studies, and Nancy J. Diment and Gloria Brown forsecretarial support.

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2. Bowry, S. C., P. N. Chhuttani, R. N. Chakravarti, and A. K.Sehgal. 1966. Study of oesophagus, stomach and duodenum of100 Rhesus monkeys. J. Assoc. Phys. India 14:235-238.

3. Bronsdon, M. A., and F. D. Schoenknecht. 1988. Campylobacterpylori isolated from the stomach of the monkey, Macacanemestrina. J. Clin. Microbiol. 26:1725-1728.

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7. Glupczynski, Y., A. Burette, M. Labbe, C. Deprez, M. DeReuck,and M. Deltenre. 1988. Campylobacter pylori-associated gastri-tis: a double-blind placebo-controlled trial with amoxycillin.Am. J. Gastroenterol. 83:365-372.

8. Glupczynski, Y., M. Delmee, C. Bruck, M. Labbe, V. Avesani,and A. Burette. 1988. Susceptibility of clinical isolates of Cam-pylobacter pylori to 24 antimicrobial and antiulcer agents. Eur.J. Epidemiol. 4:154-157.

9. Glupczynski, Y., M. Labbe, and A. Burette. 1987. Treatmentfailure of ofloxacin in Campylobacterpyloridis infection. Lanceti:1096.

10. Goodwin, C. S., J. A. Armstrong, and B. J. Marshall. 1986.Campylobacter pyloridis, gastritis, and peptic ulceration. J.Clin. Pathol. 39:353-365.

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