GASTRIC SHIGELLOSIS IN RHESUS MONKEYS

THOMAS H. KENT, M.D.,* SAMUEL B. FORMAL, PH.D., E. H. LABEEC, PH.D.,H. SPRINZ, M.D., AND RONALD M. MAENZA, M.D.

From the Departments of Experimental Pathology and Applied Immunology,Walter Reed Army Institute of Research, Washington, D. C.

Known types of gastritis due to specific bacteria include syphilis,tuberculosis, and spreading submucosal abscesses (phlegmonous gastri-tis) due to streptococci or, less commonly, other bacteria. These types ofgastritis are usually hematogenous in origin and probably do not in-volve the mucosa primarily. It is generally thought that primary bac-terial infections of the gastric mucosa do not occur because of the lowpH of gastric contents. There is little present-day information on thissubject, but the older German literature contains reports of gastricmucosal involvement in human dysentery.'

Lapin and Yakovleva 2 described desquamation of epithelium and in-creased mucus with occasional shallow erosions in the stomachs ofmonkeys with shigellosis, but Ogawa et al.8 found the alimentary lesionsin monkey shigellosis to be limited to the colon.

In our experiments on monkey shigellosis we have observed a severeulcerative gastritis and have demonstrated Shigella in the lesions byfluorescent antibody and cultural techniques. These findings form thebasis of this report.

MATERIAL AND METHODSObservations were on rhesus monkeys (Macaca mulatta), weighing 2-4 kg., which

had been fed approximately io 10 Shigella flexneri organisms in 20 CC. of brain-heartinfusion broth by gastric tube. Monkey chow and water were allowed ad libitum. Themonkeys were killed at various times after challenge, and the gastrointestinal tractwas removed, opened, pinned out flat, and immersed in io% formalin buffered with2% sodium acetate. Sections for histology were taken from anterior wall, lessercurvature, and posterior wall as previously described.4 Tissues for fluorescent micros-copy were frozen and processed by previously described methods.5 Swabs of luminalcontents of the stomach and intestines were streaked on MacConkey agar plates andsuspicious colonies were picked for identification.

RESULTS

During the course of experiments on monkey shigellosis we observeddiffuse gastric mucosal lesions in 5 animals with dysentery which were

Accepted for publication Apr. iI, I967.*Present address: Department of Pathology, University of Iowa College of Medicine,

Iowa City, Iowa 52240.

259

killed at 3, 4, 4, 7, and 8 days after challenge with Shigella flexneri ibor 2a. Since we had not previously systematically examined the stomachof a larger series of monkeys with shigellosis, we challenged an addi-tional I5 monkeys with S. flexneri 2a and killed them 48 hr. afterchallenge. Seven of these animals had received a killed Shigella vaccineparenterally which is ineffective in preventing shigellosis (unpublisheddata). Since there were no differences in the parenterally vaccinatedand nonvaccinated groups, they will be presented together.On gross examination at 2 days, 7 of I5 monkeys had patchy hemor-

rhage in various areas of the gastric mucosa, but most prominent at thejunctions of the body with the fundus or focally in the pylorus. At 3 or4 days the gastric body mucosa had a diffusely hemorrhagic appearance(Fig. i; compare with normal in Fig. 2 ). In fundic and pyloric mucosathe hemorrhages were more patchy or punctate. The lesions endedabruptly at the esophageal and duodenal junctions. At 7 or 8 days theappearance of the mucosa was more granular than normal and minimalhemorrhage and congestion were present.

Microscopic examination at 2 days revealed that i i of I 5 animals hadmild to moderate gastric lesions with focal to patchy involvement of thegastric body mucosa and focal involvement of the fundus and antrum insome animals. The lesions were predominantly shallow ulcerations withdegeneration of epithelial cells, polymorphonuclear leukocytic infiltra-tion, and exudation with surrounding edema and hemorrhage (Fig. 3).At 3 or 4 days the lesions were more severe, with multifocal ulcerationsextending for a variable depth into the mucosa and occasionally into thesubmucosa (Fig. 4). Interstitial edema and hemorrhage was more severethan at 2 days. The lesions were more prominent in the body but alsoinvolved the fundus and antrum. At 7 and 8 days the lesions appeared tobe mostly healed, with prominent hyperplasia of the surface and foveolarmucus-secreting epithelium, variable loss of glands, and patchy chronicinflammatory cell infiltrate. However, focal areas of ulceration were stillpresent.

In Giemsa-stained sections of stomach, large bacilli were easilydemonstrated in epithelial cells at the margins of the ulcerated areas atall time intervals (Fig. 5). The bacilli were identified as Shigella infrozen sections treated with specific Shigella antiserum tagged withfluorescien (Fig. 6).The colons of I4 of I5 animals killed at 2 days and the animals killed

at 3 and 4 days had a diffuse or patchy acute inflammatory reaction ofvariable severity. The lesions were similar to those previously describedin a large series of monkeys killed at 2 days after challenge.6 The monkeykilled at 7 days had extensive mucosal ulceration and abscesses in lymph-oid patches of the colon. The anirnal killed at 8 days had a moderate

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patchy colonic inflammation, which appeared to be in a stage of resolu-tion.The ileum of i animal killed at 2 days had a slight acute inflammatory

lesion in the mucosa overlying a Peyer's patch, but the other animalskilled at 2-4 days had a normal jejunum and ileum. The animal killed at7 days had a mildly inflamed ileum with a crypt: villus ratio of i: i in-stead of a normal ratio of I:4. The animal killed at 8 days had a severeileal lesion with polymorphonuclear leukocytes in the mucosa, bacilliin epithelial cells, and a crypt:villus ratio of 3: I .There were no significant lesions in other viscera except for mild

fatty metamorphosis of the liver.Shigella flexneri was cultured from the stomach content of IO of 15

animals killed at 2 days. Three of the negative cultures were fromanimals without gastric lesions and 2 were from animals with mildlesions. Nine of io of the positive cultures were from animals withgastric lesions. The pH of gastric contents in animals with negativecultures were i.6, I.7, i.8, 4.2, and 7.I. The gastric pH in animals withpositive cultures were i.8, 2.1, 2.5, 3.0, 3.7, 6.5, 6.7, 7.I, 7.3, and 7.4.

Shigella were cultured from the stomach of 4 of 5 animals killed at3 to 8 days. The animal with the negative culture was killed at 4 daysand had large numbers of shigellae in the gastric mucosa, as determinedby the fluorescent antibody technique. Shigellae were also cultured fromileum and colon of animals killed at 3-8 days, but cultures of heart blood,liver, spleen, and gastric and colonic lymph nodes were negative forShigella.We have carefully examined the stomachs of 132 monkeys that did

not have Skigella dysentery and have not seen a similar lesion. Thesemonkeys include I 5 challenged with various attenuated Skigella-Escherichia coli hybrids, 77 monkeys killed at i hr. to ii days afteradministration of staphylococcal enterotoxin, and 40 monkeys killedI-7 days after challenge with virulent and avirulent salmonellae.Staphylococcal enterotoxin produces an acute gastritis which does notulcerate and has much less tendency to involve that area of the stomachcontaining parietal cells.4 Monkeys given virulent salmonellae oftenhave small foci of acute inflammatory cells in various areas of thegastric mucosa which are not hemorrhagic or ulcerative.7 In a previouspaper,4 3 monkeys were described with gastric body ulcers; in retro-spect, we find that these and only these monkeys had not been protectedwith live Shigella vaccine prior to challenge with Shigella.

DIsCUSSIONWe have demonstrated the occurrence of gastric shigellosis in rhesus

monkeys with experimental dysentery induced by oral feeding of either

26IAug. z967

Shigella flexneri ib or 2a. The type and the sequence of the inflammatoryreaction in the gastric mucosa was similar to that in the colon. Bacilliwere demonstrated with Giemsa-stained sections from the lesions andidentified as Shigella in frozen sections by the fluorescent antibodytechnique. Shigellae were also cultured from the gastric contents. Theprominence of bacilli in epithelial cells, especially at the margin ofulcerated areas, is also characteristic of colonic shigellosis in monkeys6and Shigella conjunctivitis in guinea pigs.8We are not aware of any animal studies demonstrating the occurrence

of acute mucosal gastritis in which the causative bacteria have beenidentified in the lesion. LobeckI described 2 cases of necrotizing gastri-tis and 4 cases of esophagitis among 39 cases of acute dysentery in man,and demonstrated bacteria in histologic sections of the lesion. Werdt9described catarrhal gastritis in human cases of chronic dysentery andcultured dysentery bacilli from the gastric contents. Lobeck' andWerdt9 refer to other reports of gastritis in human bacillary dysentery.In light of the present findings in monkeys, further work on the roleof bacteria in acute and chronic gastritis using modern bacteriologic andhistologic techniques may be worth while.

In I937 Garrod'0 found that Shigella flexneri was more resistant togastric juice than other pathogens and postulated that "the infectivityof intestinal pathogens varies with their resistance to bactericidal actionin the stomach." Our observations substantiate the ability of Shigellaflexneri to survive in the stomach at varying pH's. However, more ex-perimental work is needed to assess the role of gastric acidity in relationto acute infections and to determine quantitatively the ability of variousorganisms to survive in the stomach under defined conditions.The role of acid in the production of gastric shigellosis in monkeys

is not clear, although it seems unlikely that the shigellae are meresecondary invaders in lesions produced by excess acid. Gastric andduodenal ulcers have been produced by psychological stress in mon-keys,11'12 but our monkeys were not stressed to the degree reported asnecessary to produce ulcers in monkeys. Furthermore, monkeys sim-ilarly handled but challenged with Shigella-E. coli hybrids, salmonellae,or staphylococcal enterotoxin did not develop ulcers. The type of ulcersdescribed in stressed animals were usually localized and present in eitherstomach or duodenum. In contrast, the ulcers in monkey shigellosis aremore diffuse and are not present in the duodenum.

SUMMARYTwenty rhesus monkeys were fed virulent Shigella flexneri organisms

and the stomachs were examined at 2, 3, 4, 7, and 8 days after challenge.

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Grossly nearly half, and microscopically more than two-thirds of theanimals had gastric lesions as early as 2 days post challenge. Theselesions were most severe at 3-4 days, but persisted to 8 days. Of 20 an-imals, i6 had gastric lesions while i9 had the colonic lesions of shigel-losis. In Giemsa-stained sections large bacilli were demonstrated in thelesions. The fluorescent antibody technique specifically identified thesebacteria as shigella. Shigella flexneri was cultured from the stomachs ofI3 of the I6 animals with gastric lesions. There appeared to be no re-lationship between pH of gastric contents and the occurrence of gastriclesions or the recovery of Shigella organisms from gastric contents.

REFERENCESi. LOBECK, E. Ueber nekrotisierende Osophagitis und Gastritis bei Bazillenruhr.

Zbl. Allg. Path., 33:206-2I7, I923.2. LAPIN, B. A., and YAKOVLEVA, L. A. Comparative Pathology in Monkeys.

Thomas Publisher, Springfield, Ill., I963.3. OGAWA, H., TAKAHASHI, R., HONJO, S., TAKASAKA, M., FuJIwARA, T., ANDO, K.,

NAKAGAWA, M., MUTO, T., and IMAIZuMI, K. Shigellosis in Cynomologusmonkeys (Macaca irus) III. Histopathological studies on natural and experi-mental Shigellosis. lap J Med Sci Biol z7:32I-332, I964.

4. KENT, T. H. Staphylococcal enterotoxin gastroenteritis in rhesus monkeys.Amer I Path 48:387-407, I966.

5. LABREc, E. H., SCHNEIDER, H., MAGNANI, T. J., and FORMAL, S. B. Epithelialcell penetration as an essential step in the pathogenesis of bacillary dysentery.J Bact 88:1503-15I8, I964.

6. FORMAL, S. B., KENT, T. H., AUSTIN, S., and LABREc, E. H. Fluorescent anti-body and histological study of vaccinated and control monkeys challengedwith Shigella flexneri. J Bact 91:2368-2376, I966.

7. KENT, T. H., FORMAL, S. B., and LABREc, E. H. Salmonella gastroenteritis inrhesus monkeys. Arch Path (Chicago) 82:272-279, I966.

8. FORMAL, S. B., LABREc, E. H., KENT, T. H., and FALKOW, S. Abortive intesti-nal infection with an Escherichia coli-Shigella flexneri hybrid strain. J Bact89:I374-I382, I965.

9. VON WERDT, F. Die pathologische Anatomie der chronischen Ruhr. I. Diemakroskopisch erkennbaren Veranderungen. Frankfurt Z Path 28:379-406,I922.

IO. GARROD, L. P. The susceptibility of different bacteria to destruction in thestomach. (Abst.) J Path Bact 45:473-474, 1937.

II. PORTER, R. W., BRADY, J. V., CONRAD, D., MASON, J. W., GAAMBOS, R., andRIOCH, D. M. Some experimental observations on gastrointestinal lesionsin behaviorally conditioned monkeys. Psychosom Med 20:379-394, 1958.

12. BRADY, J. V., PORTER, R. W., CONRAD, D. G., and MASON, J. W. Avoidance be-havior and the development of gastroduodenal ulcers. I Exp AnaW Behav z:69-72, 1958.

[Illustrations follow]

KENT ET AL.

LEGENDS FOR FIGuREsFIG. I. Gastric shigellosis 4 days after challenge with S. flexneri 2a. Body mucosa

is diffusely hemorrhagic. Lesion is more patchy in fundus and punctate in antrum.Esophagus (top) and duodenum (bottom) are not involved.

FIG. 2. Normal monkey stomach.FiG. 3. Early ulcerative lesion of gastric body mucosa 2 days after challenge with

S. flexneri 2a, demonstrating epithelial degeneration, polymorphonuclear leuko-cytic infiltrate, and surface exudate. Hematoxylin and eosin stain. X I35.

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FIG. 4. Ulcer of gastric body 3 days after challenge with S. flexneri lb. Hematoxylinand eosin stain. X I30.

FIG. 5. High power of margin of ulcer in Fig. 3, demonstrating bacilli in epithelialcells and lamina propria. Giemsa stain. X goo.

FIG. 6. Frozen section of ulcerative gastric body lesion treated with fluorescein-labeled S. flexneri 2a antiserum. Specifically fluorescing organisms are presentin lumen and in tissue of ulcerated area. X 525.

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