EMERGING INFECTIOUS DISEASES
Volume 1, Number 4, October-December 1995

DISPATCH

(Pages 115-23)

Helicobacter hepaticus, a Recently Recognized Bacterial Pathogen,
Associated with Chronic Hepatitis and Hepatocellular Neoplasia in
Laboratory Mice

Gastric carcinoma, one of the most prevalent human cancers
worldwide, is among the neoplasms for which epidemiologic
evidence of environmental causes is strongest. The exact nature
of these environmental causes was obscure until mounting evidence
recently linked chronic infection of the gastric antrum mucosa by
Helicobacter pylori (a microaerobic, gram-negative, spiral
bacterium) with elevated cancer risk (1). It is now recognized
that gastric B-cell lymphoma of mucosa-associated lymphoid tissue
is also closely linked to gastric H. pylori infection, and
eradication of the infection with antibiotics can result in
regression of the lymphoma (2,3). This startling finding has
stimulated intense interest in the genus Helicobacter and related
organisms; as a result, additional species of Helicobacter are
now frequently isolated and characterized from many non-human
hosts. Until 1994, however, only H. pylori was known to be
associated with tumor development, in humans or in any other
animal species.

In 1992, at the National Cancer Institute's Frederick Cancer
Research and Development Center (FCRDC) in Frederick, Maryland, a
high prevalence of liver disease was observed among certain
strains of mice; these mice were untreated controls in long-term
chemical carcinogenesis experiments. Affected strains, notably
A/JCr, had been bred at FCRDC under pathogen-free conditions and
were free of known serologically detectable murine viruses and
parasites; moreover, they had no histologically demonstrable
hepatic abnormalities, except for a very low incidence (1% to 2%)
of hepatocellular tumors in mice 15 months of age or older. Over
a very short period, the prevalence of a histologically
distinctive form of hepatitis increased to virtually 100% in male
mice at 1 year of age (Table 1). The earliest demonstrable
lesions were small, undistinctive foci of hepatic necrosis seen
in young mice aged 2 to 6 months. In older mice, aged 6 to 10
months, there was a highly distinctive pericholangitis,
consisting of abundant mononuclear cell infiltrates around bile
ducts within portal triads. The biliary epithelium within
affected ducts was focally swollen, and the luminal surfaces of
damaged epithelial cells were poorly defined in hematoxylin and
eosin-stained sections (4,5). In livers with extensive lesions,
bile ductular (oval cell) hyperplasia was also prominent.
Moreover, mice with hepatitis usually had hepatocellular tumors,
often multiple, that included both adenomas and carcinomas (4).

Hepatocellular tumors in mice are one of the most common
endpoints in bioassays for chemical carcinogens. They were not,
at that time, known to be associated with infectious agents.
Accordingly, initial efforts to identify the cause of the
hepatitis/hepatocellular tumor syndrome were directed toward
possible sources of chemical exposure. The possibility of
accidental exposure to experimental substances within the
research animal facilities was ruled out when liver disease was
identified in mice that had never left the breeding areas which
are located in separate buildings. Extensive chemical analyses of
food, bedding, water, and other possible sources of toxic
substances had negative results.

Detailed pathologic examination by light microscopy of tissue
sections from diseased livers was continued, and many special
stains were used. One such stain, Steiner's silver impregnation
procedure for spirochetes (6), revealed in hepatic tissue uniform
bodies that were consistent in size and shape with bacteria.
Homogenates of fresh liver tissue from diseased mice proved
effective in transmitting hepatitis to A/J mice purchased from
commercial sources outside FCRDC, when given by intraperitoneal
injection (5). In addition, from these homogenates, a motile,
spiral bacterium could be cultivated on blood agar plates
incubated at 37 degrees C under anaerobic or microaerobic
conditions.

This organism was subsequently characterized by ultrastructural
morphologic examination, biochemical characteristics, and 16S
rRNA gene sequence. Determined to be a new species related to H.
pylori, it was given the name H. hepaticus (7). The bacterium is
motile and gram negative, 0.2 to 0.3 m in diameter, 1.5 to 5.0
m long, and curved to spiral in shape, with one to several
spirals; it has bipolar sheathed flagella (one at each end) but
lacks the periplasmic fibers that envelope the bacterial cells in
other mouse Helicobacter species. H. hepaticus has strong urease
activity, is oxidase and catalase positive, produces H2S, reduces
nitrate to nitrite, and grows microaerobically at 37 degrees C
but not at 25 degrees C or 42 degrees C. It is resistant to
cephalothin and nalidixic acid but sensitive to metronidazole.
Photographs illustrating its morphologic structure by light (4,5)
and electron (4,5,7) microscopy have been published. The
species-defining characteristic of the organism, the nucleotide
sequence of its 16S rRNA gene, has been used to develop a
diagnostic assay based on polymerase chain reaction (8).

Systematic examination of rodents of all species and strains
produced at FCRDC, especially retired breeders, showed that the
characteristic hepatitis and associated bacteria were present in
mice of several strains (A/JCr, DBA/2NCr, C3H/HeNCr) and that
within these strains, the male mice were more severely affected
than the female. Mice with severe combined immunodeficiencies
were especially vulnerable. The precise location of organisms
demonstrable by Steiner stain within infected liver parenchyma
was shown by transmission electron microscopy to be invariably
extracellular and characteristically within bile canaliculi
(4,5). No liver disease was seen in some strains (e.g.,
C57BL/6NCr) or in F1 hybrids between sensitive and resistant
strains (e.g., B6C3F1). Rodent species other than mice (e.g.,
rats, Syrian hamsters, and guinea pigs) were not affected.

In infected mice with severe combined immunodeficiency, cecal
inflammation was histologically demonstrable (5), and organisms
were isolated from the mucosa of the large intestine (7), which
may mean that the usual ecologic niche occupied by H. hepaticus
is that of a commensal colonizer of the intestinal tract (8).
Since mice are coprophagic, it appears highly likely that natural
transmission of the organisms is the oral-fecal route. Why and
how H. hepaticus invades the liver in mice of certain strains
remain to be determined. Hepatitis is also characteristic of
certain other enteric pathogenic bacteria, such as Campylobacter
jejuni (9) that, unlike H. hepaticus, have not been associated
with liver tumor development. The tissue damage that accompanies
persistent infection by H. hepaticus, H. pylori, and certain
other Helicobacter species may be due, at least in part, to a
soluble, trypsin-sensitive cytotoxin of high molecular weight
produced by these organisms (10). There is no precedent for any
direct role of such a toxin in carcinogenesis. On the other hand,
chronic infections by viruses, bacteria, or certain parasites are
recognized risk factors for human cancers at various sites. The
hypothesis that chemically reactive, potentially genotoxic,
substances of low molecular weight (including nitric oxide and
active oxygen species) generated by inflammatory cells at the
site of chronic infection may initiate or enhance carcinogenesis
has been examined (11). The hypothesis is under active
investigation in the context of H. hepaticus-associated liver
disease.

H. hepaticus is susceptible to a number of antibiotics; treatment
of susceptible, naturally infected 8- to 10-week-old strain A/JCr
mice with single or combined antimicrobial agents has been
evaluated for efficacy in eradicating established infections
(12). Amoxicillin, metronidazole, and tetracycline administered
singly failed to eradicate bacteria from the gastrointestinal
tract, but either amoxicillin or tetracycline, in combination
with metronidazole and bismuth, was effective in eradicating H.
hepaticus from the liver, cecum, and colon when given by oral
gavage for a period of 2 weeks (12). The effect of antibiotic
therapy on the carcinogenic process, or in older animals, remains
to be established.

The importance of H. hepaticus to humans is not yet completely
known. The organism clearly has the potential to confound
bioassays for chemical carcinogens, but this potential has no
direct effect on humans. Even though most Helicobacter species
identified to date are characteristically associated with (and
named after) specific mammalian host species in which they
generally inhabit the gastrointestinal tract (with or without
causing gastritis or other chronic inflammatory disease), the
potential host range for some species is quite broad. H. pylori,
originally isolated from humans, has recently been isolated also
from the domestic cat; this raises the possibility that
Helicobacter pylori may be a zoonotic pathogen that can be
transmitted from companion animals to humans (13). Exploring the
possibility of zoonotic transmission of H. pylori, H. hepaticus,
or any other Helicobacter species would require isolation of the
organism in question by culture methods. Serologic methods have
not yet been refined to the level of species specificity. Humans
infected with H. pylori mount a serum antibody response to the
bacteria that is readily detected by enzyme-linked immunosorbent
assays and is considered evidence of ongoing disease (1); mice
infected with H. hepaticus similarly produce serum antibodies to
that species that have been demonstrated by Western blotting (5).
Antisera to H. pylori can be used to visualize H. hepaticus in
mouse liver tissue sections stained by the avidin-biotin complex
immunohistochemical procedure (5). The cross- reactivity between
these two species precludes use of available serologic methods to
establish whether H. hepaticus has infected humans.

Regardless of whether H. hepaticus is itself capable of infecting
humans, it serves to demonstrate that liver tissue can be
persistently infected by at least one member of the genus
Helicobacter, and that liver cancer can be a long-term
consequence of such infection. This discovery raises questions
about the existence of a comparable relationship between liver
cancer in humans and unrecognized bacterial infections. Reviews
are under way of tissue blocks from pathology archives in search
of organisms demonstrable by the Steiner stain in liver sections
from human populations at high risk for liver cancer.

Jerry M. Rice
Laboratory of Comparative Carcinogenesis, National Cancer
Institute, Frederick, Maryland, USA


Table 1. Increasing prevalence of hepatitis and hepatocellular 
neoplasia in control male A/JCr mice at the National Cancer 
Institute's Frederick Cancer Research and Development Center, 
1989-1992a    

               Number    Age,      Mice with       Mice with
Date killed    of mice   in weeks  hepatitis (%)   liver tumors (%)
___________________________________________________________________

Jan-Mar 1989     48       47+or3     0                 0

May-Jul 1989     47       70+or60    0                 1 (2)

Jan-Apr 1992      6       36+or4     2 (33)            0

Jul 1992         16       54         16 (100)           1 (6)

Aug-Oct 1992      6       64+or3     5 (83)            3 (50)

Dec 1992         12       77         12 (100)          11 (92)
______________________________________________________________
(a)Adapted from ref. 4.

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