Vol. 58, No. 6

INFECTION AND IMMUNITY, June 1990, p. 1763-1768

0019-9567/90/061763-06$02.00/0 Copyright © 1990, American Society for Microbiology

Inoculation of Barrier-Born Pigs with Helicobacter pylori: Animal Model for Gastritis Type B L. ENGSTRAND,1* S. GUSTAVSSON,2 A. JORGENSEN,3 A. SCHWAN,'

a

Useful

A. SCHEYNIUS4 and Surgery,2 University Hospital, and The National Veterinary Institute,3 AND

Departments of Clinical Bacteriology' Uppsala, and Department of Clinical Immunology, Karolinska Institute, Stockholm,4 Sweden Received 4 December 1989/Accepted 14 March 1990

At the age of 8 weeks, 15 barrier-born pigs, specific pathogen free, were inoculated intragastrically with suspensions of 107 to 1010 CFU of Helicobacter pyloni after pretreatment with omeprazole. The pigs were observed for up to 12 weeks, endoscopic biopsy specimens were taken, and serum samples were drawn. H. pylori was identified by routine culturing and by staining with an H. pylon-specific monoclonal antibody on cryostat sections of gastric biopsy specimens. In 11 of 15 inoculated pigs, H. pyloni was detected throughout the observation period. In these infected pigs, there was an antibody response to H. pylori, as determined in serum by an enzyme immunoassay. Furthermore, the development of superficial, focal gastritis with infiltrates of mononuclear class II antigen-expressing lymphocytes was observed immunohistologically. H. pyloni was never detected and an antibody response to H. pyloni was not observed in two control pigs. The development of gastritis and the systemic antibody response to H. pyloni support the usefulness of this animal model for studies of H. pylon-related human diseases.

Since the initial reports of Marshall and Warren in 1983 (16, 26), a large number of investigators have agreed that the gram-negative spiral bacterium Helicobacter pylori (7), until recently known as Campylobacter pylori, is an important candidate agent for the genesis of gastritis and peptic ulcers (1, 3, 8, 20). As with many human diseases, the understanding of H. pylori gastritis would increase if an animal model of this infection were available for experimental use. Oral challenge with H. pylori in numerous small laboratory animals like mice, rats, rabbits, and guinea pigs has, so far, been unsuccessful (4). Since a recent study indicated that pigs might be suitable animals for the study of the characteristics and pathological mechanisms of H. pyloriassociated gastritis (14), we decided to investigate whether barrier-born pigs could serve as an animal model for H. pylori infections. The aims of the present experiments were to establish the growth of H. pylori in the gastric mucosae of pigs, to elucidate local inflammatory and systemic immune responses, and to try to clear the organism from the infected mucosae.

MATERIALS AND METHODS Animals. Seventeen Swedish Landrace barrier-born pigs from four litters were studied (see Table 1). Barrier-born pigs are free from the specific pathogens affecting the individual herds. The animals used in the present study are free from the following common infectious agents in pigs: Mycoplasma spp., Bordetella spp., Haemophilus spp., Treponema spp., Campylobacter spp., Brucella spp., Leptospira spp., Pasteurella multocida, pathogenic Escherichia coli, Salmonella spp., Clostridium perfringens, and Mycobacterium spp. Such pigs may harbor Corynebacterium pyogenes, beta-hemolytic streptococci, and coagulase-positive staphylococci but without signs of clinical infections. At the age of 8 weeks (body weight, 15 to 20 kg), the pigs were transferred to ordinary experimental sties and the inoculation was started. The pigs were kept isolated from other pigs and given ordinary pig feed. *

Corresponding author.

Bacterial preparation. A strain of H. pylori was isolated from a 43-year-old male with a long history of peptic ulcer disease. The strain was identified by Gram staining and by indirect immunofluorescence with an H. pylori-specific mouse monoclonal antibody (MAb) (5). It was urease, catalase, and oxidase positive and negative for nitrate reductase. The bacteria were grown on blood agar plates at 37°C in anaerobic jars with the CampyPak system (BBL Microbiology Systems, Cockeysville, Md.). The colonies were harvested after 4 days and suspended in sterile 0.9% saline at room temperature. The suspension was administered within 45 min, and part of it was cultured on blood agar plates as a control. Inoculation and observation. Fifteen pigs were inoculated at gastroscopy (see below), and 2 served as controls. Acid secretion was inhibited by oral pretreatment with omeprazole (40 mg daily) for 1 week before the inoculation of H. pylori. All pigs were fasted for 14 h before inoculation and were given intravenous papaverine (40 mg) 5 min before inoculation. The inoculation program varied as follows. The five pigs in the first litter received a suspension of 5 x 108 CFU in 5 ml of 0.9% sterile saline at weeks 0 and 1 (Table 1). The three pigs in the second litter were inoculated three times with a suspension of 109 CFU in 5 ml of 0.9% sterile saline (see Table 1). In this litter, 20 ml of intragastric gruel was administered before and after administration of the bacterial suspension, followed by 50 ml of fat (Intralipid; Kabi Vitrum, Stockholm, Sweden) injected into the duodenum via the endoscope to delay gastric emptying. The four pigs in the third litter received a suspension of 5 x 107 CFU in 5 ml of 0.9% sterile saline at weeks 0 and 1 (see Table 1). A third inoculation of 3 x 108 CFU at week 2 was administered with gruel and fat as described above. Three pigs in the fourth litter were inoculated once with a suspension of 9 x 109 CFU in 10 ml of 0.9% sterile saline together with gruel and fat as described above (see Table 1). Two controls from litter four received 10 ml of 0.9% saline and were kept separated from the infected pigs. Endoscopy was performed weekly for the first 3 weeks and then every other week. In litters 1 and 2 serum samples were drawn at weeks 0 and 6, 1763

1764

ENGSTRAND ET AL.

and in litters 3 and 4 serum samples were drawn at week 0 and then every other week (see Fig. 4). Antibacterial treatment. Ten weeks after the inoculation of H. pylori, the four infected pigs from the third litter were treated with a combination of bismuth subsalicylate (500 mg twice daily), metronidazole (750 mg twice daily), and bacampicillin (800 mg twice daily) for 2 weeks. The drugs were administered orally 1 h before meals with a 20-ml syringe. Endoscopy and biopsy specimens. Prior to endoscopy, the pigs were fasted with free access to water for 18 to 24 h. To diminish coprophagia, we replaced the water with a glucose solution 10 h before endoscopy. Endoscopy was performed with azaperonum (4 mg/kg)-metomidatichlorid (20 mg/kg) anesthesia and an Olympus GIF-Q endoscope. The esophageal, gastric, and most of the duodenal mucosae were examined systematically without knowledge of previous culture results. Two biopsy specimens were taken from the prepyloric area (within 2 to 5 cm from the pylorus). One specimen was snap frozen in chilled isopentane and stored at -70°C for subsequent histopathological and immunohistochemical staining (see below). The other specimen was homogenized and cultured for H. pylori (see below). Culturing of H. pyloni. The biopsy samples were homogenized and inoculated on blood agar plates with and without antibiotics as described elsewhere (6). Suspected colonies were Gram stained. The H. pylori isolates were always identified as described above. Histopathology. Histopathological assessments of the specimens from litters 3 and 4 were performed on 6-,um-thick cryostat sections stained with Mayer hematoxylin-eosin. The locations and numbers of lymphocytes and plasma cells in the epithelium and in the lamina propria were noted. Immunohistochemical staining. Cryostat sections, 6 ,um thick, were fixed in acetone for 5 min at 4°C. The following antibodies were used: a MAb specific for H. pylori and denoted E7C11 (5) (undiluted); a MAb, ISCR 3, reactive with the swine major histocompatibility complex class II antigens, termed the SLA class II (DRw) antigens (27) (diluted 1/100); a MAb directed against the p-chain of human major histocompatibility complex class II antigens, cross-reactive with the SLA class II antigens, and denoted 4-24-11 (L. Akerblom, unpublished data; provided by A. Osterhaus, Rijks Instituut Woor Volksgezondheit en Milieuhigiene, Bilthoven, Holland) (diluted 1/20); and a rabbit antiserum directed against human class II antigens (12) and crossreactive with the SLA class II antigens (diluted 1/20). The peroxidase-antiperoxidase method (25) was used to visualize binding of the MAbs. Rabbit anti-mouse immunoglobulins (diluted 1/40) used as a secondary antibody and preformed complexes of horseradish peroxidase and mouse monoclonal anti-horseradish peroxidase antibody were obtained from Dakopatts, Copenhagen, Denmark. The peroxidase reaction was developed with 3-amino-9-ethylcarbazole (11), and the sections were counterstained with Mayer hematoxylin. Binding of the rabbit anti-class II antibodies was investigated by alkaline phosphatase-catalyzed staining (18). In this case, the sections were preincubated with normal swine serum (diluted 1/10), and swine anti-rabbit immunoglobulins (diluted 1/50; Dakopatts) were used as the secondary antibody followed by incubation steps with rabbit anti-alkaline phosphatase (diluted 1/200) prepared as described earlier (18) and alkaline phosphatase type VII (10 ,ug/ml; Sigma Chemical Co., St. Louis, Mo.). The alkaline phosphatase reaction was developed in naphthol-AS-MX buffer (Sigma) containing 1 mg of fast-red TR salt (Sigma) per ml, and the sections were counterstained with Mayer hematoxylin. Controls without

INFECT. IMMUN.

the primary antibodies did not stain. The optimal dilutions of the antibodies were determined with sections from lymph nodes obtained from one pig (no. 12) in litter 3 at the end of the experiments. Serum immune response. Just before gastroscopy, serum samples were collected and frozen at -20°C for the subsequent determination of H. pylori antibodies. Sera were tested for the development of antibodies by a conventional enzyme immunoassay (23). The bacterial strain used for inoculation was used as the coating antigen (100 p.l of a 107-CFU/ml suspension of H. pylori was added to each well). The serum samples were diluted 1:10 and further in twofold dilutions up to 10,240. Peroxidase-conjugated rabbit antiswine immunoglobulins (Dakopatts) were used as the secondary reagent. The reaction was measured in a Titertek Multiscan photometer (Flow Laboratories, Hearts, England) at 495 nm. Preimmune sera from all pigs were included in each assay. RESULTS Clinical signs. No clinical signs of disability such as diarrhea, vomiting, weight loss, or poor appetite were noted in any pig during the observation period. Detection of H. pylon. H. pylori could be cultured from 11 of 15 inoculated pigs (Table 1). Once the pigs were infected with H. pylori, the bacteria could be cultured from the pigs throughout the observation period (Table 1). Immunohistochemical staining with the H. pylori-specific MAb E7C11 was performed in the infected pigs from litters 3 and 4 (Table 1). No bacteria were observed in culture-negative pigs, but in 80% of infected (culture-positive) pigs H. pylori could clearly be detected by positive immunoperoxidase staining, size, morphology, and location (Fig. 1). Endoscopy observations. Endoscopy is technically easy to perform in anesthetized pigs, but sometimes the endoscopic view was obscured by gastric contents. In normal pigs, the gastric mucosa of the corpus is reddened, i.e., below the obviously squamous cell epithelial third proximal part. This reddened area of the corpus is sharply demarcated from the pale antrum. In the H. pylori-infected pigs, the reddened area of the corpus extended further distally and, typically, the red areas encircled pale areas, giving the mucosa a patchy appearance. These findings were observed in all infected pigs 1 to 3 weeks after inoculation of the bacteria in parallel with the detection of H. pylori by culturing. In the immediate prepyloric area or in the duodenum changes were never observed, nor did obvious erosive gastritis or ulceration develop. No macroscopic mucosal abnormality was observed in the noninfected or control pigs. Histological and immunohistochemical observations. Before inoculation, occasional mononuclear leukocytes were present in the lamina propria and in the epithelial regions (Fig. 2). In the H. pylori-infected pigs, the number of mononuclear lymphocytes increased 2 to 6 weeks after inoculation. The mononuclear lymphocytes were located in the lamina propria and in the epithelium and sometimes in aggregates (Fig. 3A and B). There was no deep infiltration through the submucosa. The histological picture in the H. pylori-infected pigs varied and was most pronounced from weeks 4 to 8. The number of mononuclear cells and aggregates tended to decrease towards the end of the observation period (after week 8) in all infected pigs, i.e., the picture became nearly identical to that seen before inoculation. In the noninfected pigs and controls, histological changes were not observed during the 8-week observation period.

VOL. 58, 1990

H. PYLORI-ASSOCIATED GASTRITIS IN BARRIER-BORN PIGLETS

1765

TABLE 1. Detection of H. pylori in antral biopsy specimens from pigs inoculated with H. pylori and from noninoculated controls Litter

Culture (immunoperoxidase staining') result at wk:

Pig no.

ob 1^

2e

3ej

49

2b

lb

3

4

1

-

-

-

-

ND"d

2 3 4 5

-

-

-

-

-

-

+

+ +

+ + +

ND ND ND ND

-

+ + +

+ + +

ND

+(+) +(-) -(-)

+

-

6 7 8

9 10 11 12 13 14 15 16 17

-

-

-

-

ND ND +

6

8

10

12

+ + +

ND

ND

+ + +

+ + +

+ + + +

+

+

+

+ +

+()

+

+ + +

-

-

-

-

-

-

-

-

-

- (

+

+(+)

+

-

-

-

-

+

ND

+(

)

++-

+-

+

-

a Immunoperoxidase staining was done with a MAb specific for H. pylori. b Serum samples were drawn and biopsy specimens were taken before inoculation was started. c Inoculated with H. pylori at weeks 0 and 1. d ND, Not done. eInoculated with H. pylori at weeks 0, 1, and 2. f All pigs in litter 3 were treated from weeks 10 to 12. g Pigs no. 13 through 15 were inoculated with H. pylori at week 0.

The mononuclear cells described above expressed class II antigens when stained immunohistochemically (Fig. 2 and 3A and B). Similar results were obtained with MAb ISCR 3, MAb 4-24-11, and the rabbit antiserum against human class II antigens. Expression of class II antigens was not found on the epithelial cells in any biopsy specimen. Serological results. Raised antibody titers to H. pylori were detected in sera from all 11 H. pylori-infected pigs. In litters 1 and 2, a twofold increase in antibody titers was demonstrated 6 weeks after inoculation. Antibody responses in litters 3 and 4 could be detected 2 to 4 weeks after challenge, and the titers remained high throughout the observation period (Fig. 4). Controls and pigs challenged but not infected had no antibody response specific for H. pylori. Antibacterial treatment. Two weeks of combination therapy cleared H. pylori from the mucosa in all treated pigs (as determined by an absence of growth; Table 1). Antibody titers remained elevated 2 weeks after clearance (Fig. 4). Histological examination of the biopsy specimens after clearance (at week 12) showed a distribution and numbers of mononuclear cells similar to those seen before treatment at week 10. The macroscopic appearance was not changed after treatment and showed reddened areas as described above.

DISCUSSION A nonexpensive, easily reproducible, and convenient animal model of H. pylori infection is urgently needed. The present study shows that barrier-born pigs are susceptible to gastric infection with H. pylori, as demonstrated by repeated cultures and immunohistochemical detection of H. pylori, systemic antibody responses, endoscopic signs of slight gastritis, histopathological findings of local inflammatory responses and, finally, clearance of H. pylori after treatment. Our experiments indicate that a high oral inoculation dose

and repeated inoculations enhance the infection rate. The doses used in the present study are comparable to those used in a human inoculation experiment (17) and in the gnotobiotic piglet model (14). Our hypothesis is that hypochlorhydria and retarded gastric emptying should make the stomach more susceptible to colonization with H. pylori, but these assumptions must be proved in controlled experiments. To assess the suitability of an animal in a model for human H. pylori infections, some considerations must be made. The argument in favor of pigs is that their gastrointestinal system is anatomically and physiologically similar to that of humans. Furthermore, a recent study described a gastric glycerolipid that was detected in both human and pig stomach tissues and that was specifically recognized by isolates of H. pylori and proposed to be a specific receptor for H. pylori on the gastric mucosa (15). Concerning the normal microbial flora of the pig stomach, some information is available (24). Barrier-born pigs are specific pathogen free, i.e., they should not harbor certain infectious agents. When we cultured for H. pylori in the antrum of the pig stomach, we found several gram-negative and gram-positive rods, such as Klebsiella spp., E. coli, Pseudomonas spp., diphtheroids, and lactobacilli, i.e., these pigs were far from gnotobiotic. Naturally occurring gastritis and ulceration in pigs have been described (22). There is a difference between ulcers occurring in glandular and nonglandular areas of the pig stomach. The nonglandular area surrounds the cardia and is frequently the site of ulceration. Only quite recently have authors distinguished between ulcers of the pars oesophagea (nonglandular region) and those of the glandular gastric mucosa. The latter are primarily seen in the fundic region but are sometimes accompanied by ulceration of the glandless esophageal area. The etiology of these peptic ulcers still needs to be clarified, but some causative agents have been

1766

INFECT. IMMUN.

ENGSTRAND ET AL.

g,

Ax

AIf *44j~~1.4W

44k

FIG. 1. Immunoperoxidase staining of H. pylori with MAb E7Cll (arrow) on a cryostat section of a gastric biopsy specimen from an infected pig (no. 11). The section was counterstained with Mayer hematoxylin.

FIG. 2. Immunoperoxidase staining of a cryostat section of a pig gastric mucosa before inoculation of H. pylori. Mononuclear leukocytes expressing class II antigens (stained by MAb 4-24-11) are indicated by arrows. The section was counterstained with Mayer hematoxylin.

proposed (not H. pylori, however) (13). Few, if any, clinical signs may be seen, and the ulcers are only found at slaugh-

antra of the

ter.

Experimental models for gastric ulcer production in pigs have been reported. Increases in gastric acid and pepsin, secretion by secretagogs like histamine (19), and administration of ulcerogenic drugs (9) all lead to ulceration of the gastric mucosa. Naturally occurring gastritis of the glandular region has been described but differs from human gastritis by its location along the greater curvature. Its pathogenesis is poorly understood. Physiologic hyperemia, hemorrhage, infarction, and erosion may be observed but are nonspecific. The lesions are considered merely to reflect an enteric disease or septicemic process (10). In our experiments, the macroscopic lesions were superficial and located in the regions in which H. pylori was present. The development of obvious erosive gastritis or ulceration was never observed, perhaps because of the short period of infection. Gastritis in pigs is not classified as in humans. The histopathological lesions in the present study were comparable to those seen in a human focal superficial gastritis. Similar findings have been described in the gnotobiotic pig model (14), in which neutrophilic infiltrates were also found, mainly restricted to the nonglandular area 1 week after challenge. This area was not studied in our experiments. Neutrophils, typically found in human gastritis type

B (28), were not observed as a component of gastritis in the

pigs.

We have recently described the induction of expression of class II transplantation antigens on epithelial cells and an increased number of T lymphocytes in gastric biopsy specimens in human H. pylori-associated gastritis (6). The expression of class II antigens on epithelial cells was not observed in the infected pigs, but there was an increase in class II antigen-expressing mononuclear lymphocytes both in the epithelium and in the lamina propria, indicating a local cellular response. The histological changes in our study diminished towards the end of the observation period, but H. pylori could still be detected in cultures. We can only speculate on the reason for this. The infection may have been self-limiting because of host defense mechanisms, as described in a human experiment (17). The specific antibody response to H. pylori after inoculation in the present study indicates that the infection is indeed a true infection and not merely a colonization. Such an antibody response in humans with H. pylor infections is well documented (21). H. pylori could not be isolated from the infected pigs after antibacterial treatment. Whether this was due to the treatment remains to be elucidated, as the infection may have been self-limiting. However, the infected pigs in litter 2 and the only infected pig in litter 4 remained infected for 12

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Inoculation of barrier-born pigs with Helicobacter pylori: a useful animal model for gastritis type B.

At the age of 8 weeks, 15 barrier-born pigs, specific pathogen free, were inoculated intragastrically with suspensions of 10(7) to 10(10) CFU of Helic...
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