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Previous Article | Next Article 
Infection and Immunity, April 2000, p. 2110-2118, Vol. 68, No. 4
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Chronic Atrophic Gastritis in SCID Mice
Experimentally Infected with Campylobacter fetus
Vincent B.
Young,1,2
Charles A.
Dangler,3
James G.
Fox,1,3 and
David B.
Schauer1,3,*
Division of Bioengineering and Environmental
Health1 and Division of Comparative
Medicine,3 Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, and Infectious
Diseases Unit, Department of Medicine, Massachusetts General
Hospital, Boston, Massachusetts 021142
Received 27 September 1999/Returned for modification 30 November
1999/Accepted 13 January 2000
 |
ABSTRACT |
Campylobacter fetus is a cause of enteritis and
invasive extraintestinal disease in humans. In order to develop an
animal model of C. fetus infection, outbred ICR SCID mice
were orally challenged with a clinical isolate of C. fetus.
The stomachs of SCID mice were heavily colonized with C. fetus, and colonization was associated with the development of
chronic atrophic gastritis. This lesion was characterized by an
inflammatory infiltrate of granulocytes and macrophages that over time
resulted in a loss of specialized parietal and chief cells in the
corpus and the appearance of a metaplastic mucous epithelium. This
lesion bears similarity to that encountered during experimental murine
infection with Helicobacter pylori or Helicobacter
felis. Despite colonization of the cecum and colon tissues by
C. fetus in SCID mice, no lesions were noted in these
tissues. A follow-up study confirmed these findings for SCID mice and
also demonstrated that C. fetus could also infect the
gastric mucosa of wild-type, outbred ICR mice. However, in ICR mice,
the anatomic extent of colonization was more limited and the severity
of inflammation and epithelial alterations was significantly less than
that observed in infected SCID mice. The stomach may represent an
unrecognized environmental niche for Campylobacter species.
| |
INTRODUCTION |
Campylobacter species are
important causes of enteritis and invasive disease in humans. In the
United States, C. jejuni and C. coli are the most
common causes of bacterial enteritis (3). C. fetus subsp. fetus (referred to as C. fetus)
is also a cause of enteritis but is generally isolated as a cause of
bacteremia and invasive extraintestinal infections in patients with
underlying disease. It has been associated with septic arthritis,
mycotic aneurysms, endocarditis, septic thrombophlebitis, salpingitis, spontaneous bacterial peritonitis, meningitis, and cholecystitis (8). The prevalence of C. fetus enteritis is
probably underestimated because many strains do not grow well under the
selective culture conditions developed specifically for the isolation
of C. jejuni and C. coli from stool (2,
8).
Experimental infection of ferrets and nonhuman primates by C. jejuni and C. coli can result in acute enteritis
(15, 27). In addition, a number of mouse models of infection
with Campylobacter species have been described. A major
shortcoming of many of the murine models is the failure to reproduce
the most common symptom encountered in human infections, namely,
enteritis. A number of models that result in stable colonization of
mice with C. jejuni, without the development of diarrhea,
have been introduced. They have been used to demonstrate the importance
of several colonization factors in establishment of mucosal
colonization (10, 25). In addition, colonization models have
been used to aid in the development of potential vaccines against
C. jejuni (1, 4, 6). In other models,
pretreatment of mice with iron gives lethality as a measurable end
point following C. fetus infection (24). Intranasal challenge with C. jejuni also results in
measurable lethality, but this is not the usual route of infection
among mammals (5).
Immunocompromised mice have been challenged with
Campylobacter species. Athymic germfree mice are
consistently colonized with C. fetus, without observable
mortality or morbidity (33, 34). At necropsy, the
gastrointestinal tracts of infected mice are grossly normal and no
specific histopathologic lesions are observed. Conversely, athymic
germfree mice challenged with C. jejuni develop clinical
enteritis and inflammation of the lower gastrointestinal tract
(33, 34).
The previous studies used germfree mice, which are known to have
altered expression of mucosal antigens that may result in a different
environmental niche being presented to the challenge microorganisms as
well as the absence of competition from resident microbiota
(13). In fact, when a normal fecal microbiota was introduced
to ex-germfree mice monoassociated with Campylobacter, the
Campylobacter species could no longer be cultured from the feces (33). This is in contrast to the majority of studies, in which mice with a normal fecal microbiota are persistently colonized
with C. jejuni (1, 7, 25). In the present study we extended the previous studies of Campylobacter infection
in immunocompromised mice by challenging outbred SCID mice colonized with a normal fecal microbiota with fresh clinical isolates of Campylobacter species.
| |
MATERIALS AND METHODS |
Animals and housing.
Four-week old Tac:Icr:Ha(ICR) and
Tac:Icr:Ha(ICR) scid/scid (severe combined immunodeficient
[SCID]) mice, free of murine pathogens including all
Helicobacter species, were obtained from Taconic Farms
(Germantown, N.Y.). A pilot experiment was performed with male and
female SCID mice. As similar results were obtained for both sexes, male
animals were used in the follow-up experiment. Mice were housed in an
Association for Assessment and Accreditation of Laboratory Animal
Care-approved facility in groups of five animals, separated by sex, in
sterile polycarbonate microisolator cages. For SCID animals, all food,
water, and bedding were autoclaved. All experiments were approved by
the MIT Animal Care and Use Committee.
Bacterial strains and culture conditions.
Campylobacter species used in this study were isolated
during a clinical study designed to characterize microaerobic spiral bacteria isolated from clinical stool samples (Young, Ferraro, Kachoris, Murtagh, Dewhirst, and Schauer, submitted for publication). C. fetus strain MGH 97-3574 was isolated from a human
immunodeficiency virus (HIV)-infected patient who presented with an
acute episode of enteritis manifested as 2 weeks of diarrhea and fever.
C. fetus strain MGH 97-2126 was isolated from a patient with
acute diarrhea and no underlying disease. C. hyointestinalis
strain MGH 97-2652 was isolated from an HIV-infected patient who
presented with an acute diarrheal illness. Species level identification
of these strains was based on routine biochemical characterization
(including catalase, oxidase, and urease activity, hippurate and
indoxyl acetate hydrolysis, and sensitivity to cephalothin and
nalidixic acid) and was confirmed by determination of the complete 16S
rRNA gene sequence.
After minimal passage (less than five passages) on tryptic soy agar
(TSA) supplemented with 5% sheep blood, bacteria were stored at
70°C in tryptic soy broth with 40% glycerol.
Campylobacter species were grown at 37°C in a microaerobic
environment which was maintained in vented GasPak jars without a
catalyst by evacuation to 20 mm Hg and then repressurization with a
gas mixture consisting of 80% N2, 10% H2, and
10% CO2 to yield a final O2 concentration of
5% (17).
For infection studies, bacteria were harvested after 48 h of
growth and resuspended in a small volume of tryptic soy broth. The
optical density at 600 nm (OD600) of the inoculum was
measured. Tenfold serial dilutions of the inoculum were plated to
quantify the CFU used for infection.
Experimental infection.
In a pilot infection study, five
male and five female SCID mice were challenged with C. fetus
isolates MGH 97-3574 and MGH 97-2126 and C. hyointestinalis
isolate MGH 97-2652, which were administered as a mixed inoculum. Prior
to inoculation, it was determined that the three different bacterial
strains could be distinguished on the basis of randomly amplified
polymorphic DNA (RAPD)-PCR. As controls, five male and five female SCID
mice were inoculated with sterile broth only. In a follow-up
experiment, 20 immunocompetent ICR mice and 20 immunocompromised SCID
mice were challenged orally with C. fetus strain MGH
97-3574.
Mice were inoculated with a suspension of bacteria with an
OD600 of 1.0 (~108 CFU) in a volume of 0.2 to
0.3 ml. For the pilot study involving mixed infection with C. fetus strains MGH 97-3574 and MGH 97-2126 and C. hyointestinalis strain MGH 97-2652, approximately equal numbers of
each strain were combined into a single inoculum with an
OD600 of 1.0. Bacteria were introduced directly into the
stomach with a 24-gauge ball-tipped gavage needle. Mice were challenged with a total of three equal doses of bacteria on three alternating days. An equal number of control mice were inoculated with sterile tryptic soy broth.
Necropsy and histopathology.
At 1 week, 3 weeks, and 3 months in the initial experiment and at 1, 3, and 6 months in the
follow-up study, mice were euthanized by CO2 asphyxiation.
The stomach, the liver, and small and large bowel were fixed in 10%
neutral buffered formalin and processed for histopathologic evaluation.
After 24 h of fixation in formalin, tissue samples were processed
and embedded in paraffin, sectioned at 5 µm, and stained with
hematoxylin-eosin and by the Warthin-Starry method. Sections of the
gastrointestinal mucosa were examined histologically for inflammatory
and epithelial changes and for the presence of argyrophilic spiral
organisms. Epithelial hyperplasia, atrophy (loss of normal corpus gland
morphology), and inflammation were graded semiquantitatively as 0 (normal), 1 (minimal), 2 (mild), 3 (moderate), or 4 (marked) based on
the extent and intensity of the alteration.
Microbiological culture.
A single, fresh fecal pellet was
collected from each mouse for culture. Feces were homogenized in 0.5 ml
of phosphate-buffered saline, and 50 µl was plated on TSA
supplemented with 5% sheep blood and 20 µg of cefoperazone, 10 µg
of vancomycin, and 2 µg of amphotericin B/ml (TSA-CVA).
Portions of the antrum of the stomach, the distal ileum, the cecum, and
the mid-colon were collected for culture at necropsy. The tissue was
rinsed in sterile phosphate-buffered saline, and the mucosal surface
was applied to a TSA-CVA plate. After application of the mucosal
surface to the surface of the agar, the tissue was discarded, and a
sterile, disposable inoculating loop was used to spread the sample over
the surface of the plate.
Detection of spiral organisms in feces by multiplex PCR.
DNA
was extracted from feces and subjected to analysis using a multiplex
PCR assay as described previously (Young et al., submitted). This assay
can detect and discriminate between bacteria belonging to the genera
Arcobacter, Campylobacter, and
Helicobacter.
RAPD-PCR.
RAPD-PCR was performed using the primer
5'-CAATCGCCGT-3' as previously described (19).
Statistics.
Histologic scores for the different experimental
groups were compared by nonparametric methods (Mann-Whitney U test)
using StatView, version 5.0, for Macintosh (SAS Institute Inc., Cary, N.C.).
Nucleotide sequence accession numbers.
The nucleotide
sequences of the 16S rRNA genes of the clinical isolates described in
this study have been entered in the GenBank database under accession
numbers AF219233 (MGH 97-2126), AF219234 (MGH 97-3574), and AF219245
(MGH 97-2652).
| |
RESULTS |
Campylobacter infection of SCID mice results in
persistent colonization and the development of gastritis.
To
establish an SCID mouse model of infection with
Campylobacter species, a pilot study was undertaken with
low-passage clinical isolates. Animals were challenged with a mixed
inoculum of two C. fetus isolates and one C. hyointestinalis isolate.
All mice challenged with the mixed inoculum of Campylobacter
species shed microaerobic spiral bacteria in their feces throughout the
3 months of the experiment. Three independent bacterial isolates from
each mouse were analyzed by RAPD-PCR. All isolated organisms were
identified as C. fetus strain MGH 97-3574. C. fetus strain MGH 97-2126 and C. hyointestinalis strain
MGH 97-2652 were never isolated from any of the mice after challenge
with the mixed inoculum. Multiplex PCR of DNA extracted from the feces
of infected mice detected Campylobacter at all time points
(data not shown). No Helicobacter or Arcobacter
was detected in these animals. PCR and culture for microaerobic spiral
organisms were negative in all control animals at all time points.
Throughout the experiment, none of the animals displayed any clinical
signs of disease. No diarrhea was noted, and there was no mortality
among the infected or control animals.
At all time points microaerobic spiral organisms could be cultured from
the stomach, distal ileum, cecum and colon tissues of infected animals
at necropsy. PCR of these tissues was positive for
Campylobacter, and RAPD-PCR of organisms obtained by culture demonstrated that these organisms were C. fetus MGH 97-3574.
At all time points the liver, small intestine, cecum, and colon tissues
of infected mice were histologically similar to those of uninfected
controls. Marked lesions in the stomach tissue of infected mice were
noted. One week postinfection (p.i.), spiral organisms in the pyloric
region were noted, with accompanying hyperplasia and inflammation.
Three weeks p.i., there was expansion of the area of colonization
proximally to involve the antrum, again with hyperplasia and
inflammation. By 3 months p.i. there was further expansion of
colonization with organisms found in the corpus. Hyperplasia and
inflammation now extended into the corpus as well as the antrum.
Additionally, the corpus tissue of infected animals was notable for the
development of an atrophic gastritis in which local inflammation was
accompanied by loss of specialized parietal and chief cells and the
appearance of mucous metaplasia (data not shown).
C. fetus strain MGH 97-3574 colonizes both ICR and SCID
mice.
To confirm the finding of atrophic gastritis in mice
challenged with a mixture of Campylobacter species where
C. fetus strain MGH 97-3574 was the only apparent successful
colonizer, a follow-up study using this C. fetus strain was
performed. For this experiment, both wild-type ICR and SCID mice were
infected to determine the influence of the adaptive immune system on
the development of gastritis.
One week p.i., all mice (ICR and SCID) that had been challenged were
culture positive for microaerobic spiral organisms. RAPD-PCR fingerprints of randomly selected isolated colonies matched the fingerprint of the input C. fetus strain (data not shown).
All infected animals continued to shed C. fetus for the
duration of the experiment. Multiplex PCR on feces from infected
animals detected only Campylobacter species. No evidence of
colonization with Helicobacter or Arcobacter was
ever found (data not shown). Throughout the experiment, none of the
animals displayed any clinical signs of disease. No diarrhea was noted,
and there was no mortality among the infected or control animals.
Control animals were negative for microaerobic spiral bacteria by
culture and PCR throughout the study.
C. fetus strain MGH 97-3574 causes chronic atrophic
gastritis in SCID mice.
One month after infection, the stomachs of
four of five SCID mice infected with C. fetus were notable
for hyperplasia and inflammation involving the antrum. Warthin-Starry
silver stain revealed the presence of large numbers of spiral bacteria
in the mucosa of the pylorus and distal antrum of SCID mice with
hyperplasia and inflammation (Fig. 1A and
B). The inflammatory infiltrate consisted primarily of granulocytes
accompanied by macrophages and involved both the mucosa and submucosa
(Fig. 2). No inflammation or hyperplasia
was seen in the corpus mucosa 1 month after infection (Fig.
3).
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FIG. 1.
Colonization of the stomach tissue of ICR and SCID mice
with C. fetus. Shown are low-power (A) and high-power (B)
views of the pylorus of an ICR mouse 1 month after infection with
C. fetus. Numerous argyrophilic spiral bacteria (arrows) are
localized in the antral mucosa at the level of the pyloric sphincter.
(C) High-power view of spiral organisms in the mucosa from the corpus
of a SCID mouse 3 months after infection with C. fetus. The
normal glandular zone has been replaced by a mucous epithelium.
Warthin-Starry stain. Bar, 200 µm.
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FIG. 2.
Antral inflammation in a SCID mouse 1 month after
infection with C. fetus. (A) Low-power view of the antral
mucosa demonstrating both mucosal and submucosal inflammation. (B)
Higher-power view of the antral mucosa with an inflammatory infiltrate
composed primarily of granulocytes. Hematoxylin and eoson stain. Bar,
200 µm.
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FIG. 3.
Histopathologic scoring of stomach lesions in ICR and
SCID mice infected with C. fetus. Five animals in each
experimental group were necropsied 1, 3, and 6 months after infection.
Each animal was graded for the degree of hyperplasia and inflammation
in the antrum and for inflammation and mucosal alterations (loss of
parietal and chief cells and mucous metaplasia) in the corpus.
Experimental groups were compared by the Mann-Whitney U test.
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Three months after infection, the gastritis in SCID mice had
progressed. All infected SCID mice had marked antral hyperplasia and
inflammation (Fig. 4), and the mucosa
remained heavily colonized with argyrophilic spiral organisms. The
region of colonization at 3 months expanded proximally from the pylorus
to include the entire antrum and the antrum/corpus transitional zone.
Additionally, in two of five infected SCID mice, colonization of the
corpus was seen (Fig. 1C). In these two mice, there was loss of
parietal and chief cells, with replacement by a hyperplastic and
metaplastic mucous epithelium and a brisk submucosal and mucosal
inflammatory infiltrate composed primarily of granulocytes with
macrophages (Fig. 5). This was the same
chronic atrophic gastritis lesion that was observed during the pilot
study at 3 months p.i.
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FIG. 4.
Antral inflammation and hyperplasia in ICR and SCID mice
3 months after infection with C. fetus. The antral mucosa of
an infected SCID mouse (A) is markedly hyperplastic compared to that of
an uninfected control (C). A predominantly granulocytic inflammatory
infiltrate accompanied by macrophages is present. The antrum from an
infected ICR mouse (B) has a lesser degree of inflammation and
hyperplasia than that observed in infected SCID mice. The antrum from
an uninfected control ICR mouse (D) is shown for comparison.
Hematoxylin and eosin stain. Bar, 200 µm.
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FIG. 5.
Corpus gastritis in ICR and SCID mice 3 months after
infection with C. fetus. The mucosa from the corpus of an
infected SCID mouse (A) is markedly hyperplastic compared to that of an
uninfected control (C). The mucosa and submucosa contain a
predominantly granulocytic inflammatory infiltrate accompanied by
macrophages. There is marked loss of parietal cells and chief cells and
replacement by a metaplastic mucous epithelium. An infected ICR mouse
that had mild corpus colonization with spiral bacteria (B) has a mild,
focal, submucosal inflammatory infiltrate. Widespread loss of parietal
cells is not seen, although chief cells are not prominent. The overall
mucosal height is similar to that of an uninfected control (D).
Hematoxylin and eosin stain. Bar, 200 µm.
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By 6 months after infection, four of five infected SCID mice had
extensive corpus colonization with C. fetus and associated gastritis. There was further progression of the epithelial alterations originally observed 3 months p.i. Chronic atrophic gastritis persisted, but the mucosal hyperplasia that was predominant at 3 months was no
longer apparent. Instead, there was a reduction in total mucosal height
at 6 months p.i. (Fig. 6). Other mucosal
lesions included vascular congestion in the superficial mucosa, focal
erosions of the surface epithelium, and minute hemorrhages into the
lamina propria. These changes coincided with the gross observation of dark, granular material consistent with partially digested blood in the
gastric luminal contents apposed to the mucosa. No frank mucosal
ulcerations were seen. All infected SCID mice, including the one animal
without significant corpus gastritis, had marked hyperplasia and
inflammation involving the antrum (Fig. 3). Only limited antral
inflammation and hyperplasia were observed in uninfected control mice,
significantly less than that observed in infected animals (Fig. 3). No
corpus gastritis was observed in control mice, and organisms were not
observed in gastric sections from control animals.
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FIG. 6.
Corpus gastritis in ICR and SCID mice 6 months after
infection with C. fetus. The mucosa from the corpus of an
infected SCID mouse (A) shows continued inflammation and loss of
parietal and chief cells. A metaplastic mucous epithelium remains, but
the hypertrophy seen at 3 months is no longer seen, and instead there
is a decrease in mucosal height. Infected ICR mice had focal areas of
monocytic inflammatory infiltrate (B). However, similar lesions were
also seen in uninfected control ICR mice. The severe mucosal
alterations with loss of specialized cells and mucous metaplasia were
not seen in infected ICR mice. Hematoxylin and eosin stain. Bar, 200 µm.
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Warthin-Starry staining of gastric sections from infected SCID mice
revealed the presence of large numbers of bacteria, packing many of the
glands (Fig. 1C). Some of these organisms appeared to be in close
apposition to cells. Electron microscopy confirmed the presence of
large numbers of curved bacteria in the gastric glands (Fig.
7). Although bacteria were observed in
close proximity to the apical surface of host cells, no intimate
adherence to or alteration in the ultrastructure of host cells was
seen.
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FIG. 7.
Transmission electron micrograph of infected gastric
gland from a SCID mouse 6 months after infection with C. fetus. Large numbers of curved bacteria are seen in the lumen of
the gland. The ultrastructure of the epithelial cells is unaltered.
Although bacteria are in close proximity to apical surfaces of the
epithelial cells, no intimate adherence is observed. Bar, 200 nm.
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In contrast, 1 month after infection with C. fetus strain
MGH 97-3574, three of five immunocompetent ICR mice exhibited
colonization confined to the pyloric region that was visible by
Warthin-Starry staining. Mild mucosal hyperplasia and inflammation were
also present and tended to be less severe than that in infected SCID mice, although this difference did not reach statistical significance (Fig. 3). The inflammatory infiltrate in the ICR mice was composed of
scattered foci of mononuclear cells without an active, granulocytic component.
Three months after infection, the ICR mice remained colonized in the
distal antrum and pylorus of the stomach. The expansion of colonization
to the proximal antrum that was observed in the SCID mice was not seen
in ICR mice at this time point. Mild mucosal hyperplasia and
inflammation persisted in the distal antrum but did not progress to the
same degree as that observed in the SCID mice (Fig. 4). The degree of
antral hyperplasia and inflammation in infected animals was not
significantly greater than that in controls and was much less than that
encountered in infected SCID animals at 3 months (Fig. 3). Two infected
ICR mice did have foci of spiral bacteria in the mucosa of the corpus.
These bacteria were located in the squamocolumnar transition zone (the
"cardia equivalent" of the murine stomach). In these mice, there
was moderate focal, submucosal inflammation, with a minor component of
mucosal inflammation (Fig. 5). However, the widespread loss of parietal and chief cells that was observed in infected SCID mice was not seen in
these ICR mice.
Six months p.i., all infected ICR mice remained colonized in the distal
antrum and pylorus. In three animals, colonization extended proximally
to involve that the mid-antrum. Minor inflammation and hyperplasia in
the antrum persisted in infected ICR mice but remained significantly
less than that observed in infected SCID mice (Fig. 3). Four of five
infected ICR mice were noted to have scattered foci of lymphocytic
submucosal inflammation involving the corpus (Fig. 6). The significance
of these foci of inflammation is unclear, as similar lesions were found
in control mice (Fig. 3). Small numbers of spiral bacteria were seen in
the squamocolumnar transition zone of one infected ICR mouse. Minor
epithelial alterations were seen in isolated regions of the corpus,
manifested as focal loss of parietal cells and focal regions of mucous
metaplasia. The widespread atrophic gastritis observed in the majority
of infected SCID mice was not encountered in any ICR mice by 6 months after infection.
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DISCUSSION |
We describe a novel murine model of persistent infection with
C. fetus that is characterized by gastric colonization and
the development of chronic atrophic gastritis. To our knowledge, this is the first report of experimental Campylobacter-induced
gastritis. Previous studies on germfree, athymic mice demonstrated that
both C. jejuni and C. fetus are able to colonize
the stomach, but no gastric lesions were noted (33, 34). In
humans, a clear relationship between Helicobacter pylori
infections and chronic gastritis, peptic ulcer disease, and gastric
cancer has been established (9). Campylobacter
species, however, are not generally thought to colonize the stomachs of
humans. Campylobacter species do not possess urease
activity, which has been shown to be required for gastric colonization
by H. pylori in a mouse model (21, 30). C. jejuni has been cultured from a gastric biopsy of a patient with
indomethacin-associated gastritis (28) who did not have evidence of H. pylori infection. It is not clear if this
represents primary gastric colonization with C. jejuni and
subsequent gastritis or secondary colonization of a preexisting gastric lesion.
Campylobacter species are generally thought to colonize the
lower gastrointestinal tract in humans. Although we could isolate C. fetus from the ileum, cecum, and colon tissues of
infected mice in the present study, no lesions were noted in these
tissues. Lesions in both immunocompetent ICR mice and immunocompromised SCID mice were limited to the stomach. The gastric lesions seen in SCID
mice after infection with C. fetus were particularly
striking. These mice developed severe, chronic, active gastric
inflammation that involved the entire antrum and by 6 months had
progressed to involve the corpus of the majority of animals.
Colonization and inflammation were associated with alterations in the
cytology and architecture of the gastric mucosa. In the antrum this
consisted of hyperplasia, and in the corpus this consisted initially of hyperplasia but progressed to atrophic gastritis, characterized by loss
of parietal cells and chief cells and replacement by a metaplastic
mucous epithelium. Over time, areas of overall mucosal atrophy
developed in the corpus, with reduction of mucosal thickness and
multifocal disruption of the surface epithelium. Frank mucosal ulceration was not seen however.
In both ICR and SCID mice, C. fetus initially colonized the
antroduodenal transition zone (31). Over the 6-month time
period of the study colonization expanded anatomically to a greater
degree in SCID mice than in ICR mice. In animals that developed
inflammation and mucosal changes in the corpus of the stomach, the
gastric glands were colonized with bacteria. This expansion of
colonization and inflammation mimics what has been described during
long-term human infection with H. pylori (31). It
is proposed that H. pylori initially colonizes the
antrum-body transition zone, resulting in chronic inflammation and
mucosal damage that eventually cause a decrease in local acid levels
(31). This damage and changes in the local microenvironment
at the proximal antrum-body transition zone allow the bacteria to
colonize more-proximal portions of the stomach and thus induce a moving
front of inflammation (31).
The above model can be used to explain the pathology seen in
immunocompetent ICR mice and immunocompromised SCID mice in the present
study. It is possible that ICR mice are able to mount an immune
response in the setting of C. fetus infection, dependent on
B and/or T cells, that is able to limit the extent of infection but
that cannot eliminate the pathogen. There is little damage to the
mucosa in infected ICR mice, and the bacteria remain localized to the
initial area of colonization, which appears to be the antroduodenal transition zone. In addition, the transition zone between the glandular
and squamous portions of the stomach (the cardia equivalent) may
represent a second area permissive for colonization, similar to
H. pylori colonization in humans (18). In SCID
animals, colonization with C. fetus leads to the generation
of a chronic, active inflammatory response composed chiefly of
granulocytes and macrophages. We hypothesize that this immune response
is also unable to eliminate the infection; however, it does lead to
damage of the gastric mucosa including a loss of parietal cells. This
mucosal damage leads to an expansion of the area of gastric mucosa that
can support colonization by C. fetus, presumably via a
decrease in local acid levels due to a loss of parietal cell mass. The
end result is a progression of infection from the antroduodenal
transition zone through the antrum to the corpus of the stomach.
Mice have been employed as model systems for infection by the gastric
Helicobacter species H. pylori and H. felis. Different strains of mice exhibit varied histopathologic
responses to infection with Helicobacter (14, 22, 23,
29, 32). Certain strains, exemplified by C57BL/6 mice, mount an
intense inflammatory response to infection with H. pylori or
H. felis (16, 22). These mice also develop marked
histological changes in the corpus mucosa consisting of loss of
specialized cells and the appearance of mucous metaplasia. Other mouse
strains, exemplified by BALB/c mice, although chronically colonized by
Helicobacter, develop minimal gastric inflammation, and
marked alterations in the gastric mucosa are not encountered
(29). Instead, after long-term (>22-month) infection, these
mice reportedly develop low-grade gastric MALToma-like lesions
(12).
The changes in epithelial cytology and architecture encountered in
corpus tissue of SCID mice infected with C. fetus in the present study are similar to those noted in C57BL/6 mice infected with
gastric Helicobacter. It is important to note that the mice used in the present study were regularly screened for
Helicobacter species and that none were found prior to
challenge or during the course of the study. In spite of their
similarity to the lesions that develop in the corpus tissue of C57BL/6
mice infected with H. pylori or H. felis, the
lesions that develop in SCID mice infected with C. fetus
clearly occur in the setting of a fundamentally different immune
response and are not dependent on an adaptive immune response. It has
become apparent that the gastritis and epithelial changes encountered
in C57BL/6 mice infected with gastric Helicobacter are
dependent on cellular, probably T-cell-mediated, immune responses.
Infection of C57BL/6 mice that were either RAG-1
/ or
compound T-cell receptor 
/ with H. felis resulted in high levels of colonization but no detectable
gastric pathology (26). The importance of T-cell-mediated immunity was underscored by the fact that infection of B-cell-deficient µMT mice with H. felis still resulted in gastritis and
epithelial changes (26). In another study, SCID mice on a
C57BL/6 background did not develop gastritis when colonized with
H. pylori unless they received adoptively transferred
splenocytes from C57BL/6 donor animals (11). Splenocytes
from infected or uninfected donor mice induced severe gastritis and
also suppressed bacterial colonization. In the present study, expansion
of colonization and chronic active inflammation with corpus atrophy and
mucous cell metaplasia occurred in the setting of infection with
C. fetus without the presence of T or B cells. In fact,
C. fetus infection of animals with an intact immune system
resulted in more-limited colonization and minimal active inflammation.
The development of atrophic gastritis, characterized by loss of
specialized parietal and chief cells with the appearance of mucous
metaplasia, in SCID mice suggests that these changes are the end result
of chronic gastric inflammation. It has been previously suggested that
these epithelial lesions are dependent on the presence of inflammation
and that, by themselves, even large numbers of bacteria in close
contact with the gastric epithelium are unable to cause these lesions
in the absence of a host response (11). Our results support
this hypothesis and further suggest that these epithelial lesions can
occur in the setting of non-antigen-specific (T-cell-independent)
inflammatory responses. Additionally, our electron microscope results
suggest that intimate adherence or contact between C. fetus
and host cells may not be a prerequisite for inflammation and mucosal damage.
The finding of gastritis in ICR and SCID mice infected with C. fetus was unexpected; the current system was developed to model the lower intestinal tract disease that characterizes most
Campylobacter infection in humans. During the course of the
present study, it was reported that infection of C.B-17 scid
beige mice with fresh clinical isolates of C. jejuni
resulted in the development of diarrhea in about 10% of the infected
mice (20). Histopathologic analysis of mice with diarrhea
revealed the presence of marked colonic inflammation, and no gastric
pathology was noted. It is likely that the different results seen in
this study and in our present study are due to differences in the
genetic background of the mice (inbred BALB/c versus outbred ICR)
carrying the scid mutation. Alternately, there may be
differences between the pathogenesis of C. jejuni infections
and that of C. fetus infections. However, during the pilot
infection study presented here, we also infected a group of SCID mice
with a fresh clinical isolate of C. jejuni. Similar to what
was found for mice infected with C. fetus, we noted gastric
colonization with the development of antral gastritis. No lower-bowel
lesions were seen in SCID mice infected with C. jejuni (data
not shown).
Our findings suggest that the stomach may represent an unrecognized
environmental niche within the gastrointestinal tract for
Campylobacter species. Furthermore, this system may yield additional insight into the role chronic inflammation plays in the
development of gastric pathology during infection with microaerobic spiral bacteria.
| |
ACKNOWLEDGMENTS |
This work was supported by Public Health Service grants AI01398
to V.B.Y. and by AI37750 and DK52413 from the National Institutes of Health.
We thank Floyd Dewhirst for performing 16S sequence analysis and
Kimberly A. Knox for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: MIT, Room
56-787, Cambridge, MA 02139. Phone: (617) 253-8113. Fax: (617)
258-0225. E-mail: schauer{at}mit.edu.
Editor:
R. N. Moore
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Abstract
Introduction
