Experimental Biology and Medicine
The online version of this article can be found at:
2001 226: 420Exp Biol Med (Maywood)
Dewhirst and James G. Fox
Nirah H. Shomer, Charles A. Dangler, Mark D. Schrenzel, Mark T. Whary, Shilu Xu, Yan Feng, Bruce J. Paster, Floyd E.
Species in A/J and Tac:ICR:Hascidf RF Mice
Cholangiohepatitis and Inflammatory Bowel Disease Induced by a Novel Urease-Negative Helicobacter
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Cholangiohepatitis and Inflammatory Bowel
Disease Induced by a Novel Urease-Negative
Helicobacter Species in A/J and
NIRAH H. SHOMER,* CHARLES A. DANGLER,* MARK D. SCHRENZEL,* MARK T. WHARY,*
SHILU XU,* YAN FENG,* BRUCE J. PASTER,† FLOYD E. DEWHIRST,† AND JAMES G. FOX*,1
*Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139; †Forsyth Institute, Boston, Massachusetts 02115
Helicobacter bilis and H. hepaticus, both urease-positive intestinal
helicobacters of mice, have been shown experimentally to induce
proliferative typhlocolitis in scid mice. We recently isolated a urease-
negative Helicobacter sp. (H. sp.) that also induced proliferative
typhlocolitis in pilot studies in scid mice. To determine the pathogenic
potential of H. sp. in immunocompromised and immunocompetent
mice, 5-week old male A/J or Tac:Icr:Ha(ICR)-scidfRF mice were
inoculated by intraperitoneal (IP) injection with ∼ 3 × 107colony-
forming units (CFU) of H. sp. Mice were necropsied at various time
points postinoculation (PI). Sham-inoculated mice had no clinical,
gross, or histopathological lesions. In contrast, scid mice inoculated
IP with H. sp. had severe hemorrhagic diarrhea and decreased
weight gain at 2, 7, and 18 weeks postinoculation (PI), with severe
proliferative typhlocolitis, phlebothrombosis, and hepatitis. A/J mice
had no clinical signs, but had mild to moderate proliferative typhlo-
colitis and moderate to marked cholangiohepatitis at 7 and 24 weeks
PI. A/J mice infected with H. sp. developed robust immune re-
sponses of a predominant Th1 type. This report demonstrates that
infection with a urease-negative helicobacter can cause inflamma-
tory bowel disease (IBD) and hepatitis in scid and immunocompetent
A/J mice. These results provide a new model of IBD and cholangio-
hepatitis associated with a specific urease-negative, novel H.
[Exp Biol Med Vol. 226(5):420–428, 2001]
Key words: Helicobacter; mouse; typhlocolitis; cholangiohepatitis
ease (1–8). These organisms are of particular relevance for
three reasons. First, they may be important models impli-
cating bacterial infections in carcinogenesis. Helicobacter
hepaticus, an intestinal helicobacter that has been shown to
cause persistent hepatitis in A/JCr mice, has been linked
with hepatitis and hepatic tumors in susceptible strains of
mice, and has been proposed as a model of helicobacter-
induced tumorogenesis (1, 3, 6–9). Second, many of these
enterohepatic H. species are widespread in rodent colonies
and may significantly confound the interpretation of some
research studies (10, 11). For example, some transgenic
rodent strains that have been proposed as spontaneous mod-
els of inflammatory bowel disease (IBD) are infected with
rodent intestinal helicobacters (10) that are known to poten-
tiate the development of IBD in both unreconstituted and
CD45RBhi- reconstituted scid mice (9, 12, 13). Experimen-
tal infection with H. bilis has also been shown to induce
inflammatory bowel lesions in scid mice (9, 12). Further-
more, a mixed infection of H. bilis and the recently char-
acterized H. rodentium (14) has been associated with severe
necrotizing proliferative typhlocolitis and diarrhea in a
colony of immunodeficient mice (5). However, urease-
negative H. species, until recently, have been considered
nonpathogenic (2). Third, they may have important zoonotic
implications; recently, H. bilis, H. pullorum, and “H. rap-
pini” were identified by PCR in the gallbladders and bile of
humans with chronic cholangitis (15), and novel helicobac-
ters were identified in the livers of humans with hepatocel-
lular carcinomas (16).
The purpose of this study was to determine the patho-
genic potential of a novel urease-negative H. species in both
immunocompromised and immunocompetent mice, and to
here has been considerable interest in intestinal Heli-
cobacter spp. (H. sp.) of mice because of the asso-
ciation with hepatitis and inflammatory bowel dis-
This work was funded in part by NIH (grants R01CA67529, R01DK52413,
P01CA26731, and RR01046).
1To whom requests for reprints should be addressed at Division of Comparative
Medicine, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Build-
ing 16, Room 825C, Cambridge, MA 02139. E-mail: email@example.com
Received July 21, 2000.
Accepted December 15, 2000.
Copyright © 2001 by the Society for Experimental Biology and Medicine
420 HELICOBACTER SP. CHOLANGIOHEPATITIS AND IBD IN MICE
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determine if infection with this organism in these mice
would provide a suitable model for enterohepatic disease.
Materials and Methods
Animals. Five-week-old, male Tac:Icr:Ha(ICR)-
scidfRF scid mice were obtained from Taconic Farms (Ger-
mantown, NY). The mice had a restricted flora and were
colonized with a proprietary cocktail (altered Schaedler’s
flora) of eight anaerobic bacteria (17), plus several other
nonpathogenic bacteria. All husbandry materials for scid
mice (microisolator caging, bedding, pelleted rodent chow
[Purina Mills, Richmond, IN], and water bottles) were au-
toclaved before use; all scid mouse manipulations took
place in a biosafety cabinet. Four- to 5-week-old H. spp.-
negative, male A/J mice were obtained from a commercial
vendor (Jackson Laboratories, Bar Harbor, ME). The A/J
mice were maintained in nonautoclaved filter-top microiso-
lator caging for the duration of the experiment. The mice
were fed pelleted rodent chow (Purina Mills) and were pro-
vided water ad libitum. All mice were maintained in a fa-
cility accredited by the Association for the Assessment and
Accreditation of Laboratory Animal Care International.
Animals were maintained at a constant temperature of 70°
to 72°C, 40% to 70% humidity, with 10 to 15 air changes
per hour. All manipulations performed on the mice were
approved by the Animal Care Committee of the Massachu-
setts Institute of Technology.
Bacteria. The novel urease-negative H. sp. (MIT 96-
1001) was originally isolated from the cecum of a clinically
normal feral mouse. It was grown under microaerobic con-
ditions in vented jars containing N2,H2,and CO2(80:10:10)
at 37°C in Brucella broth supplemented with 5% fetal calf
serum. The bacteria were harvested after 24 to 48 hr of
growth, resuspended in phosphate-buffered saline (PBS),
and visualized by gram stain and phase microscopy for pu-
rity, morphology, and motility. The optical density (OD600)
was adjusted to 1.0 and 0.3 ml of this inoculum (∼3 × 107
colony-forming units [CFU]) was used for each intraperi-
toneal (IP) injection or oral dose.
Bacterial Isolation. Bacteria were cultured from fe-
cal pellets (ante mortem) or from cecal contents or ground
liver at necropsy. At scheduled intervals, pooled samples of
three to five fresh fecal pellets were obtained from each
cage of mice. Fecal pellets were suspended in 1 ml of sterile
PBS, and the resulting slurry was plated directly onto CVA
plates (cefaperazone, vancomycin, amphotericin B; Remel,
Lenexa, KS) for microaerobic isolation of the novel H. sp.
At necropsy, cecal contents were applied directly to CVA
plates. Aseptically collected liver was ground and applied
directly to CVA plates for isolation of H. sp., and to blood
agar plates for isolation of aerobic bacteria. Although
growth of H. sp. was generally evident within 5 days, plates
were maintained for 2 weeks before a determination of no
growth was made.
Electron Microscopy. The novel H. sp. was exam-
ined by electron microscopy. Cells were grown on blood
agar plates (Remel) at 37°C under microaerobic conditions
for 48 h. Then they were gently resuspended in 10 mM Tris
buffer (pH 7.4) at a concentration of approximately 108cells
per milliliter. Samples were negatively stained with 1% (w/
v) phosphotungstic acid (pH 6.5) for 20 to 30 sec and were
examined with a JEOL model JEM-1200EX transmission
electron microscope operating at 100 kV.
Genomic DNA Extraction for 16S rRNA Gene
Sequencing. Bacteria were cultured on blood agar plates
and the cells were harvested and washed twice with 1 ml of
double-distilled H2O. The pellets were suspended in STET
buffer (8% sucrose, 50 mM EDTA, 0.1% Triton X-100, and
50 mM Tris HCl, pH 8.0) and lysozyme (hen egg white,
Boehringer Mannheim Biochemicals, Indianapolis, IN) was
added to a final concentration of 3 mg/ml. The suspension
was incubated for 12 min at 37°C and was then lysed with
sodium dodecyl sulfate. RNAse A (bovine pancreas; Boeh-
ringer Mannheim) was added to a final concentration of
0.05 mg/ml and the solution was incubated for 1 hr at 37°C.
Then 0.1 volume of a 5% cetyltrimethylammonium bro-
med-0.5 M NaCl solution (Sigma Chemical, St. Louis, MO)
was added and the solution was gently mixed and incubated
at 65°C for 10 min. DNA was extracted with an equal
volume of phenol-chloroform (1:1, v/v), precipitated over-
night in 0.3 M sodium acetate with 2 volumes of absolute
ethanol at −20°C, and pelleted by centrifugation at 13,000g
for 1 hr at 4°C. The ethanol was decanted and the pellet was
air dried and suspended in distilled water.
16S rRNA Gene Sequencing. The sequence of the
16S rRNA gene of the novel urease-negative helicobacter
isolate was determined. For amplification of the 16S rRNA
cistrons, 16S rRNA gene sequencing, and 16S rRNA data
analysis, we used the methods described by Fox et al. (4).
Briefly, primers C60 and B37 (4) were used to amplify the
16S rRNA genes. The amplicons were purified and directly
sequenced by using a TAQeunce cycle sequencing kit (US
Biochemica, Cleveland, OH). The 16S rRNA gene se-
quences were entered into a program for analysis of 16S
rRNA data in Microsoft QuickBASIC for use on PC-
compatible computers and were aligned as previously de-
scribed (18). The sequence database used contains approxi-
mately 100 helicobacter, wolinella, arcobacter, and campy-
lobacter sequences and more than 900 sequences for other
bacteria. Similarity matrices were constructed from the
aligned sequences by using only those base positions for
which 90% of the strains had data and were corrected for
multiple base changes by the method of Jukes and Cantor
(19). Phylogenetic trees were constructed by using the
neighbor-joining method of Saitou and Nei (20).
ELISA for Anti-H. sp. IgG in Serum and IgA in
Feces. An outer membrane antigen preparation (OMP) of
H. sp. was obtained by methods previously described for
preparing H. hepaticus antigen (21). Briefly, H. sp. was
cultured in trypticase soy broth containing 5% fetal bovine
serum for 48 hr under microaerobic conditions as detailed
above. After three washes in PBS and examination for bac-
HELICOBACTER SP. CHOLANGIOHEPATITIS AND IBD IN MICE421
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terial contaminants using gram stain and phase microscopy,
the pellet was resuspended in 4 ml of 1% N-octyl-beta-
glucopyranoside (Sigma) for 30 min at room temperature.
Insoluble material was removed by ultracentrifugation at
100,000g for 1 hr. After dialysis against PBS for 24 hr at
4°C, supernatant protein concentration was measured by the
Lowry technique (Sigma). For serum IgG measurement, 96-
well plates were coated with 100 ?l per well of 1 ?g/ml of
H. sp. OMP protein in carbonate buffer (pH 9.6) overnight
at 4°C. Coating concentration of antigen was increased to
10 ?g/ml for measurement of IgA in fecal extracts. Bioti-
nylated secondary antibodies included goat anti-mouse IgG
(Southern Biotechnology Associates, Birmingham, AL),
?-chain-specific goat anti-mouse IgA (Sigma), and mono-
clonal rat anti-mouse antibodies produced by clones G1-6.5
and R19-15 (Pharmingen, San Diego, CA) for detecting
IgG1 and IgG2a, respectively. Incubation with extravidin
peroxidase (Sigma) was followed by ABTS substrate
(Kirkegaard and Perry Laboratories, Gaithersburg, MD) for
color development. OD development at ? ? 405 m? was
recorded by an ELISA plate reader (Dynatech MR7000,
Dynatech Laboratories, Chantilly, VA).
Sample Preparation and Analysis. Sera were
collected from all mice prior to dosing with H. sp. and then
again at necropsy, and were stored at −20°C prior to analy-
sis. Serum IgG results are reported as mean OD values at a
sample dilution of 1:100, with all samples run in triplicate.
Feces were collected from all mice prior to dosing with H.
sp. and then monthly until necropsy. Four freshly voided
fecal pellets (approximately 100 mg) were suspended in a
protease inhibitor cocktail (1 ?g/ml aprotonin, 10 ?M leu-
peptin, 3.25 ? M bestatin, and 0.2 mM 4-[2-aminoethyl]-
benzene sulfonylfluoride [Sigma] in 5% nonfat dry milk) as
previously described (1). The fecal slurry was microcentri-
fuged at 10,000 rpm (Microcentrifuge 235C, Fisher Scien-
tific, Pittsburgh, PA) for 10 min to yield supernatant for IgA
measurement. Fecal extracts were frozen at −70°C pending
analysis, and were assayed undiluted. Because of an un-
known dilution factor inherent in sample preparation, the
OD measurement of IgA specific for H. sp. in fecal extracts
was standardized against total IgA concentration of the
sample. A standard curve was generated on each ELISA plate
by applying known amounts of purified mouse IgA-? (Sigma)
precoated with ?-chain-specific sheep anti-mouse IgA.
Histology. Sections of liver and gastrointestinal tis-
sues were fixed in neutral buffered 10% formalin and in
Carnoy’s fixative. Tissues were embedded in paraffin, sec-
tioned at 5 ?m, and stained with hematoxylin and eosin
(H&E) stain for assessment of histopathology, Masson’s
trichrome stain for assessment of hepatic fibrosis, or War-
thin-Starry silver stain to visualize bacteria in tissues (for-
malin-fixed sections only).
Statistics and Analysis. Statistics reported
throughout the text are based on a two-tailed Student’s t test.
Experimental Design. Pooled feces from the mice
were analyzed by PCR and were cultured microaerobically
upon arrival to ensure that they were not colonized with any
H. sp. After 1 week of acclimatization, baseline blood
samples were obtained from each mouse. Mice were ran-
domly assigned to inoculation groups. The mice were in-
oculated IP with 0.3 ml of PBS (sham-dosed controls) or 0.3
ml of PBS containing 3 × 107CFU of the novel H. sp. Two
uninoculated scid cage-contact mice were housed with IP-
inoculated scid mice for the purpose of establishing whether
natural fecal-oral transmission of the novel H. sp. occurred.
In addition, to determine whether the IP route of infec-
tion influenced the severity of clinical signs, five scid mice
were inoculated orally with 0.3 ml PBS containing 3 × 107
CFU of H. sp. Mice were observed daily and weighed
at scheduled intervals. Mice were necropsied at time
points listed in Table I. Unless otherwise stated, all data
presented in the Results section pertains to IP-inoculated
Characterization of the Novel H. sp. Bacterial
morphology and growth characteristics. Urease-
negative, catalase- and oxidase-positive, gram-negative mo-
tile bacteria grew at 37°C under microaerobic conditions.
Colonies appeared as a thin spreading layer on blood agar
media, with a characteristic feathery iridescent leading
edge. When grown in broth culture, cells could attain
lengths exceeding 10 ? m. Cells exhibited microaerobic, but
not aerobic, growth at both 37° and 42°C.
Ultrastructure. The novel H. sp. was spiral and it
measured 0.15 by 2 to 10 ?m with three to seven spiral turns
(Fig. 1). The bacterium possessed single bipolar, sheathed
flagella, but did not have periplasmic fibers.
16S rRNA analysis. The 16S rRNA sequences deter-
mined for the initial isolate (MIT 96-1001) and from two
isolates (MIT 98-5356 and MIT 98-5357) recovered from
experimentally infected mice were entered into our data-
base, aligned, and compared with the over 100 Helicobacter
Table I. Experimental Design
Number of mice necropsied
at various time points
2 WPI7 WPI 18 WPI24 WPI
scid Oral H.sp.
scid IP H. sp.
A/J IP H. sp
Note. Control mice were sham-inoculated with PBS only. Inoculated
mice were inoculated ip or orally with the 3 × 107CFUs of H. sp.—no
animals were analyzed. WPI, weeks postinoculation.
aOne control died of anesthetic complications during blood collec-
bOne out of five animals died.
cTwo out of 10 animals necropsied early (at 2 WPI).
dFive out of 10 animals died prior to necropsy; one died immediately
prior to necropsy and was included in gross, but not histological,
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sequences in the database to determine similarity. The es-
sentially complete sequence (from base 28–1524 using
Escherichia coli numbering) was determined for three
strains: 96-1001, 98-5356, and 98-5357. A neighbor-joining
phylogenetic tree showing the consensus sequence for the
novel strains and selected helicobacters is shown in Fig. 2.
The novel H. sp. sequence fell in the helicobacter cluster,
which includes H. canis, H. bilis, H. cinaedi, H. sp. Group
8, and H. westmeadii. The three sequences were identical
and were most closely related to that of H. sp. Flexispira
Group 8 (GenBank AF047851), and “H. ulmiensis” (Gen-
Bank AJ007931). The novel H. sp. strains contain an inter-
vening sequence (IVS) that is essentially identical to that in
H. bilis and H. sp. Flexispira Group 8. The novel H. sp.
sequence differs by 12 bases from that of the “F. rappini”
(GenBank AF047851) and differs from the “H. ulmiensis”
sequence (AJ007931) by seven bases in the ribosomal por-
tion of the sequence (data not shown); however, “H. ulmien-
sis” contains a uniquely different IVS.
Clinical and Gross Findings. A/J mice. There
were no clinical signs or significant difference in the body
weight of control and H. sp.-inoculated A/J mice at any time
point (Fig. 3). Control mice had no gross lesions at necropsy
at 7 or 24 weeks PI. Multiple 1- to 5-mm pale yellow foci
in the liver parenchyma (corresponding to hepatic necrosis)
were observed in two of 10 inoculated mice at 7 weeks PI.
Translucent nodules and tracts (corresponding to mono-
nuclear cell accumulations seen by light microscopy) were
apparent on cut sections of the livers of nearly all (eight of
10) inoculated mice at 24 weeks PI.
scid mice. Control mice weighed significantly more
than the mice inoculated with the novel H. sp. at every time
point (Fig. 3). Control mice had no clinical signs and no
grossly apparent lesions at necropsy at 2, 7, 18, or 24 weeks
PI. In contrast, all mice inoculated IP with H. sp. developed
Figure 2. 16SrRNA analysis. Neighbor-
joining phylogenetic tree based on 16S
rRNA sequence comparisons (20). Scale
bar represents 5% difference in nucleo-
tide sequences, as described by measur-
ing the lengths of horizontal lines con-
necting any two species. Species names
are followed by strain number, GenBank
accession number (in curly brackets),
and animal species from which strain
Figure 1. Ultrastructure of the novel Helicobacter species. (Top)
Transmission electron micrograph (bar = 0.5 µ m). The novel H. sp.
is motile and spiral, and measures 0.15 by 2 to 10 µm. with two to
seven spiral turns. (Bottom) (bar = 0.2 µm) The bacterium has single
bipolar, sheathed flagella. but does not have periplasmic fibers.
HELICOBACTER SP. CHOLANGIOHEPATITIS AND IBD IN MICE423
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intermittent severe bloody diarrhea at 2 weeks PI. Diarrhea
episodes typically lasted 1 week, with frequent relapses
noted. Eight IP-inoculated scid mice died or were eutha-
nized by 18 weeks PI. Multiple 1- to 5-mm pale yellow foci
in the liver parenchyma (corresponding to hepatic necrosis)
were seen in five of 10 IP-inoculated mice at 7 weeks PI. At
18 weeks PI, there was gross evidence of massive hepatic
necrosis and remodeling of the livers of three of four sur-
viving H. sp.-inoculated scid mice. They had abnormally
shaped fused liver lobes indicative of prior massive necrosis
and subsequent repair.
Orally dosed scid mice. To determine if the severe
clinical signs were due to the IP route of inoculation, five
scid mice were orally inoculated with H. sp. These mice
developed bloody diarrhea within 1 week of inoculation, an
even earlier time point than in the IP inoculated scid mice,
indicating that the clinical signs were not an artifact of IP
Uninoculated scid cage contact mice. To determine
if fecal-oral transmission could occur, two uninoculated
scid cage contacts were co-housed with IP-inoculated in-
fected mice 2 weeks after inoculation. The cage contacts
also developed intermittent severe bloody diarrhea within 1
week of exposure.
Colonization by the Novel H. sp. All of the sham-
dosed scid (n ? 20) and A/J mice (n ? 20) remained
culture-negative for the novel H. sp. The H. sp.-dosed scid
and A/J mice were culture positive within 2 weeks of dosing
by the IP route. H. sp. was also isolated from the feces of
orally inoculated scid mice and the uninoculated scid cage
contacts within 2 weeks of exposure. The novel H. sp. was
readily cultured from the livers of selected IP-inoculated
scid or A/J mice with gross liver lesions. A few slender
spiral argryophilic organisms consistent with H. sp. were
seen in selected livers stained with Warthin-Starry silver
Histopathology. IP-inoculated scid mice No le-
sions were observed in the livers or gastrointestinal tracts of
the control scid mice at 7 weeks PI. At 18 weeks PI, mild
mucosal hyperplasia and limited focal inflammation were
observed in the rectums of two control mice. Mild focal
hyperplasia of the gastric antral mucosa was also observed
in two control mice at this time point. In contrast, infection
with H. sp resulted in moderate-to-marked proliferative
typhlocolitis accompanied by hepatic necrosis and inflam-
mation both mice necropsied at 2 weeks PI. At 2 weeks PI,
IP-inoculated scid mice had moderate proliferative typhlo-
colitis with multifocal epithelial necrosis. Hyperplastic
crypts were elongated and lined by densely packed, hyper-
chromic epithelium with diminished goblet cell differentia-
tion (Fig. 4a). The proximal colon and cecum were further
characterized by submucosal edema and infiltration by his-
tiocytes, granulocytes, and small mononuclear cells (Fig.
4b). Both of the mice necropsied at the 2-week time point
developed phlebothrombosis within the portal circulation,
accompanied by extensive, multifocal coagulative necrosis
and inflammation (Fig. 4c). Subacute periportal inflamma-
tion and multifocal hepatic microabscessation were promi-
nent in the affected livers.
At 7 and 18 weeks PI, moderate-to-marked prolifera-
Figure 3. Effect of intraperitoneal inoculation with the novel H. sp.
on body weight. Mean body weight of infected and control scid and
A/J mice at the indicated time points: Infected scid mice weighed
significantly less (P < 0.004) than the control mice at every PI time
point. There was no significant difference in the weights of control
and Helicobacter-infected A/J mice at any time point.
Figure 4. Histopathology of H. sp. infection in scid mice. (a) Prolif-
erative typhlitis in a scid mouse inoculated IP with H. sp. (18 weeks
PI). Characteristic features of proliferative typhlitis were observed at
2, 7, and 8 weeks PI in scid mice. Cecal hyperplasia is characterized
by focal areas of increased crypt length, with increased mucosal
epithelial cell hyperchromicity and increased crypt cell density. The
mucosal cellular infiltrate consists primarily of mononuclear cells
morphologically characterized as macrophages, with neutrophils and
occasional eosinophils. Similar lesions were seen at 7 weeks PI.
100×. (b) Proliferative colitis in a scid mouse inoculated IP with H. sp.
(2 weeks PI). (proximal colon) The colonic inflammatory infiltrate was
similar to the cecal infiltrate (Fig. 4a). At 7 and 18 weeks PI, the
proximal colon was normal, and only the distal colon and cecum
were affected. 100×. (c) Severe hepatitis with extensive coagulative
necrosis in a scid mouse inoculated IP with H. sp. (2 weeks PI).
Similar lesions were seen at all time points. Thrombi were frequently
present in veins. 40×. (d) Coagulative hepatic necrosis in a scid
mouse inoculated IP with H. sp. (18 weeks PI). A fibrous capsule
(arrow) surrounds areas of coagulative necrosis (asterisk). 400×.
424 HELICOBACTER SP. CHOLANGIOHEPATITIS AND IBD IN MICE
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tive typhlitis was consistently observed in eight out of eight
and four out of four mice, respectively (Fig. 4a). Cecal
epithelial necrosis was observed in several mice (not
shown). The intensity of colonic inflammation was dimin-
ished to mild intensity present in only seven out of eight
mice at 7 weeks PI, and colitis was manifested as moderate
hyperplasia and inflammation polarized toward the distal
colon and rectum in four out of four mice at 18 weeks PI.
Multifocal hepatic phlebothrombosis was common, associ-
ated with moderate-to-marked hepatic necrosis and inflam-
mation. Remodeling of the hepatic tissue was manifested
grossly as large, abnormally contoured lobes. Portal inflam-
mation, multifocal necrosis, fibroplasia, organization of
thrombi, and multifocal suppurative inflammation were ob-
served in hepatic tissue of eight out of eight mice at 7 weeks
PI and three out of four mice at 18 weeks PI (Fig. 4d).
Extrahepatic thrombi were also rarely observed in veins of
the cecum and pancreas in mice from the 7-weeks PI group.
IP-inoculated A/J mice. No significant lesions were
observed in the livers or gastrointestinal tracts of the control
A/J mice at 7 and 24 weeks PI. Infected A/J mice typically
developed less intense inflammation and proliferation of the
cecum and colon, as compared with scid mice. At 7 weeks
PI, proliferative typhlitis (present in 10/10 mice) was mild
in intensity, and was characterized by hyperplasia and mul-
tifocal or diffuse infiltration by lymphocytes. At 24 weeks
PI, typhlitis progressed toward mild (four out of 10 mice) or
moderate (five out of 10 mice) intensity (Fig. 5a), and in one
case developed a severe inflammation and mucosal hyper-
plasia with multifocal cecal ulceration. Moderate focal gas-
tric antral hyperplasia and inflammation and mild ileitis
were also observed in several mice. Mild-to-moderate coli-
tis was observed infrequently (three out of 10 mice), limited
primarily to the distal colon or rectum.
A unique pattern of hepatic lesions, progressing to
chronic cholangiohepatitis, developed in response to experi-
mental infection in A/J mice. At 7 weeks PI, moderate-to-
marked portal hepatitis and multifocal necrosis were ob-
served in six out of 10 mice (Fig. 5b). Multifocal hepatic
phlebitis, characterized by prominent lymphocytic perivas-
cular infiltrates, was also present. At 24 weeks PI, in eight
out of 10 mice, the portal inflammation was marked in
intensity and was manifested by large lymphoplasmacytic
infiltrates that formed thick cuffs around portal structures,
especially bile ducts (Fig. 5c). Scattered bile ducts con-
tained suppurative exudate and necrotic cells (Fig. 5d). He-
patic fibrosis was observed as a pattern of mild-to-moderate
collagen deposition, extending from the portal areas into the
Serology. Serum IgG responses. A/J mice experi-
mentally infected with the novel H. sp. developed signifi-
cant (P < 0.001) serum IgG responses to the infection by 24
weeks PI (Table II). Using an isotype-specific ELISA for
discrimination between IgG2a (Th1-like) and IgG1 (Th2-
like) antibody responses, mice infected with the novel H. sp.
developed a predominant IgG2a serum response to H. sp.
infection. The IgG2a response was significantly higher than
the IgG1 response in all mice tested (P < 0.0005) with a
mean ratio of IgG2a to IgG1 of 2.8 ± 0.6.
Fecal IgA responses. The total IgA concentration in
the fecal extract samples was equivalent across experimen-
tal groups (data not shown). The A/J mice infected with H.
sp. developed significant IgA responses compared to con-
trols by 24 weeks PI (P < 0.007; Table II).
The results of this study show that experimental infec-
tion with a novel urease-negative helicobacter, H. sp., can
cause severe proliferative typhlocolitis, hepatic phlebo-
thrombosis, and hepatitis in outbred scid mice, and moder-
ate proliferative typhlocolitis and marked cholangiohepatitis
in male A/J mice. We have previously reported an outbreak
of severe diarrhea and proliferative necrotizing ulcerative
typhlitis in inbred (C.B17) scid mice associated with a dual
infection of H. bilis and H. rodentium, (5) and moderate
typhlocolitis in female outbred ICR scid mice infected with
H. bilis (12). Others have reported mild typhlocolitis and
hepatitis in male C.B17 scid mice infected with H. bilis (9).
Several factors, including strain and sex, have been shown
to modulate the severity of helicobacter-induced disease
(22). Lesions from infection with H. hepaticus have been
Figure 5. Histopathology of novel H. sp. infection in A/J mice (a)
Proliferative typhlitis in an A/J mouse infected with H. sp. for 24
weeks. Cecal hyperplasia is characterized by focal areas of in-
creased crypt length, increased mucosal epithelial cell hyperchro-
micity, and increased crypt cell density. Cecal inflammation consist-
ing primarily of mononuclear cells with neutrophils and occasional
eosinophils was present. 100×. (b) Necrosis and hepatitis in an A/J
mouse infected with H. sp. for 7 weeks. Hepatitis, characterized by
pericholangial and perivascular inflammatory cell infiltrates and mild-
to-moderate areas of coagulative necrosis (asterisk), was present in
the majority of the Helicobacter-infected mice at 7 weeks PI. 100×.
(c) Severe cholangiohepatitis in an A/J mouse infected with the novel
H. sp. for 24 weeks. Infiltrates were concentrated around biliary
tracts, and necrosis and perivascular inflammation were minimal.
Bile duct reduplication was observed. 40×. (d) Chronic suppurative
cholangitis in an A/J mouse infected with the novel H. sp. for 24
weeks. A dense lymphoplasmacytic and histiocytic cell infiltrate sur-
rounds the bile duct, which is filled with a suppurative exudate. A
neutrophil (PMN) can be seen translocating across the bile duct wall
HELICOBACTER SP. CHOLANGIOHEPATITIS AND IBD IN MICE425
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reported to vary with mouse strain and sex. (6, 8). The
enterohepatic lesions observed in H. sp-inoculated mice in
this study were considerably more severe than those seen in
our previous pilot study, which used female defined flora
ICR scid mice (23). Also, the clinical signs in the scid mice
were so severe that the original experimental design (nec-
ropsy at 7 and 24 weeks) had to be modified (necropsy at 2,
7, and 18 weeks). It is unlikely that the severe hepatic ne-
crosis, which resulted in massive remodeling of the livers of
some mice, was simply due to the IP route of exposure, as
this route has been used in other studies (8, 23, 24) with no
similar consequences. Also, the orally exposed scid mice
and even the scid mice exposed only by cage contact de-
veloped severe lesions and developed clinical signs at an
earlier time point than the IP-inoculated mice.
Our interpretation is that the clinical signs were due to
colonization of the gut by the H. sp., and that this coloni-
zation takes place faster when the bacteria are inoculated
directly per os than when they have to reach the gut via the
IP route. The increased severity of the enterohepatic lesions
noted in mice in the current study may be due to a gender
predilection or to the presence of other bacterial species in
the gut of “restricted” rather than “defined” flora mice. Fi-
nally, there may be bacterial-specific factors that are impor-
tant determinants of the severity of disease. For instance,
strains of H. pylori that elaborate vacuolating cytotoxin
have been reported to be more pathogenic than strains with-
out the cytotoxin (25, 26). H. hepaticus has been reported to
produce a granulating cytotoxin (27) and a newly reported
cytolethal distending toxin (28). Urease is an essential colo-
nization factor for gastric helicobacters, allowing for the
organism’s survival in an acidic environment. Urease is
produced by the established enterohepatic pathogens H. he-
paticus and H. bilis, but not by the novel H. sp. reported on
here, or by “H. typhlonicus,” which has been reported to
cause disease in IL10−/−and scid mice (2, 29). Thus, our
results confirm that urease is not a required virulence factor
for enterohepatic helicobacters to cause hepatic or enteric
disease. It remains to be determined whether there are viru-
lence factors unique to the novel H. sp., or strains of the
novel H. sp. that are more or less pathogenic.
The liver lesions reported in this study are similar to
those seen in aged mice naturally infected with H. hepaticus
(8) and to IL-10−/−mice naturally or experimentally in-
fected with another recently described urease-negative H.
sp. (H. typhlonicus), which is taxonomically distinct from
the novel H. sp used in this study (2, 29). The early onset of
these lesions in our study (apparent at 7 weeks) may be due
to the route of exposure (IP versus oral) or may indicate that
the novel urease negative H. sp. is more virulent. The severe
cholangitis and pericholangitis may be the direct result of
colonization of the liver and bile ducts with H. sp. or may
reflect an autoimmune response directed against an antigen
common to intestinal helicobacters and the biliary epithe-
lium (30). Prolonged (1–2 years) infection with H. hepati-
cus is associated with the development of hepatic tumors
(31). Although the development of inflammatory lesions
was greatly accelerated compared to infection with H. he-
paticus, no hepatic tumors were seen in mice infected with
the novel H. sp. The study was of insufficient duration to
determine the carcinogenic potential of H. sp. and further
studies with longer-term infection are clearly needed. Infec-
tion with H. hepaticus also causes a significant hepatic vas-
culitis (3, 31). It has recently been theorized that H. pylori
exerts some pathogenic effects on the vasculature, and it has
been epidemiologically and anecdotally linked with vascu-
lar disorders such as migraine and Reynaud’s syndrome
The significant serum IgG and fecal IgA responses of
the A/J mice to the novel H. sp. are consistent with the
marked inflammatory response to the infection. Mice in-
fected with the novel H. sp. developed a serum IgG2a an-
tibody response that was greater then the IgG1 response,
indicating that a pro-inflammatory Th1 response to the in-
fection predominated over a Th2 response, as previously
observed in A/JCr mice infected with H. hepaticus (21) and
in C57BL/6 mice infected with H. felis (33).
It has been hypothesized that some human IBD is trig-
gered by bacterial antigens and that the IBD is a dysregu-
lated immune response to unspecified bacterial insult (34–
36). Several mouse models of IBD have been reported to
result from the elimination or overexpression of cytokines
involved in the balance between a Th1 and Th2 response
(37, 38). For example, Kullberg et al. (37) showed that H.
hepaticus induces chronic colitis in SPF-reared IL-10(−/−)
mice, and the disease is accompanied by a type 1 cytokine
response (IFN?, TNF?, and nitric oxide). In contrast, wild-
type C57BL-background animals infected with the same
Table II. Mean OD ± SE of ELISA for Serum IgG and Fecal IgA
Infection status IgGIgAIgG1IgG2a
Novel H. sp.
0.069 ± 0.009
2.056 ± 0.034a
0.092 ± 0.015
0.295 ± 0.032a
0.004 ± 0.006
0.665 ± 0.065a
0.000 ± 0.005
1.822 ± 0.133a,b
Note. Serum IgG and Fecal IgA at 24 weeks PI. Sera and fecal extracts from A/J mice (n = 10) infected for 24 weeks with the novel H. sp. were
tested in a serum IgG and fecal IgA ELISA against h. sp. antigen. Sera from a subset of five A/J mice infected for 24 weeks with the novel H.
sp. were tested in an isotype-specific ELISA for discrimination between IgG2a and IgG1 serum antibody responses. The IgG, IgA, IgG1, and
IgG2a responses were significantly elevated compared with controls and the IgG2a (Th1-like) response was significantly higher than the IgG1
(Th2-like) response in the mice infected with H. sp.
aSignificant compared to controls at p < 0.001
bSignificant compared to IgG1 response at p < 0.001
426HELICOBACTER SP. CHOLANGIOHEPATITIS AND IBD IN MICE
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bacteria did not develop disease and they produced IL-10 as
the dominant cytokine in response to Helicobacter antigen.
In vivo neutralization of IFN? or IL-12 resulted in a sig-
nificant reduction of intestinal inflammation in H. hepati-
cus-infected IL-10−/−mice, suggesting an important role for
these cytokines in the development of colitis in the model.
Apparently, in immunocompetent hosts, IL-10 stimulated in
response to intestinal flora is important in preventing IBD.
The A/J mice in this study exhibited a Th1 response to the
novel H. sp. and a robust inflammatory response in the liver.
However, the scid mice also developed severe enterohepatic
lesions, similar in quality to those in the A/J mice, despite a
lack of T cells. The inflammatory infiltrate in the scid mice
was primarily mononuclear, predominantly consisting of
cells morphologically identified as macrophages. Although
macrophage activation is enhanced by T cells, it has re-
cently been shown that H. pylori alone can stimulate mac-
rophages and induce iNOS (39). The severe histiocytic le-
sions in the intestines and livers of the scid mice in this
study certainly suggest that novel H. sp. shares this prop-
erty, although this needs to be confirmed by in vitro testing.
Von Freeden-Jeffry et al. (38) found that H. hepaticus-
infected mice mutant for both IL-7 (a macrophage activator)
and RAG-2 (IL-7/RAG-2−/−) did not develop myeloid re-
sponses or colitis whereas RAG2−/−did, indicating that IL-7
plays a critical role in exacerbating a non-T cell/non-B cell-
mediated chronic inflammatory response. Therapy with re-
combinant IL-10 protein (which inhibits antigen presenta-
tion and macrophage production of IL-1, IL-6, and TNF?)
was able to prevent the occurrence of colitis in susceptible
RAG-2(−/−)mice, suggesting a pivotal role for macrophages
(38). The present study, as well as our earlier studies using
helicobacter-infected defined flora scid mice, using B and
T-cell free scid mice, is additional evidence for an important
role for macrophages in IBD (13, 24).
There is some evidence that intestinal helicobacters
may play a role in IBD in humans; H. cinaedi (40) and H.
fennelliae (41) were first identified as agents causing proc-
titis and colitis in immunocompromised humans (41). In
this study, a novel urease-negative murine H. sp. closely
related to a cluster of enteric helicobacters by 16S rRNA
sequence analysis produced mild-to-moderate IBD in both
outbred scid mice and inbred A/J mice. Importantly, in this
study the H. sp. also caused hepatitis with cholangitis. In
some human patients with IBD, primary sclerosing cholan-
gitis is a clinically important sequelum of unknown etiol-
ogy. An animal model with similar pathological changes
could prove valuable for investigating hypotheses related to
bacterial/autoimmune etiology of this disease process.
Intestinal helicobacters may play an important role as
factors or cofactors in the development of IBD in many
species. It is particularly important to consider the murine
intestinal helicobacters as potential pathogens. Some ge-
netic mouse models of IBD may now be attributable to H.
sp. infection (10), and some long-term toxicology studies
using B6C3F1 mice have been compromised by H. sp. in-
fection (3, 31). This study both confirms the pathogenicity
of H. sp. in immunocompetent and immunocompromised
mice, and offers a potential murine model for studies of IBD
and associated liver disease.
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