Journal of Medical Microbiology (2006), 55, 1591–1595
Case Report
DOI 10.1099/jmm.0.46707-0
Enterohepatic Helicobacter species isolated from
the ileum, liver and colon of a baboon with
pancreatic islet amyloidosis
Alexis Garcı́a,1 Shilu Xu,1 Floyd E. Dewhirst,2 Prashant R. Nambiar1
and James G. Fox1
1
Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA
02139, USA
Correspondence
Alexis Garcı́a
2
[email protected]
Department of Molecular Genetics, The Forsyth Institute, Boston, MA 02115, USA
Received 3 May 2006
Accepted 27 July 2006
Microaerobic bacteria were isolated from a baboon with pancreatic islet amyloidosis and hepatitis.
Phenotypic and molecular analyses identified two distinct helicobacters. Analyses of 16S rRNA
demonstrated ‘Helicobacter macacae’ in the ileum and liver, and Helicobacter cinaedi in the
colon. To the best of the authors’ knowledge, this is the first report describing the isolation of
enterohepatic Helicobacter species from a baboon.
Introduction
Microbiological and histological studies
Enterohepatic Helicobacter species (EHS) colonize the
intestine and liver of humans and animals (Fox, 2002).
Helicobacter hepaticus, the prototype EHS, induces chronic
active hepatitis and hepatocellular carcinoma in genetically
susceptible mouse strains (Fox et al., 1994; Ihrig et al., 1999).
Selected EHS, but not Helicobacter pylori, play a role in the
pathophysiology of cholesterol gallstone formation in C57L
mice fed a lithogenic diet (Maurer et al., 2005, 2006). In
addition, Helicobacter species DNA has been identified in
bile and gall-bladder tissue of humans with chronic
cholecystitis (Fox et al., 1998). In the present study, we
describe the isolation of EHS from the ileum, liver and colon
of a baboon with pancreatic islet amyloidosis and hepatitis.
Samples that had been collected from the ileum, caecum,
colon, gall bladder, bile and liver, and placed in culture
media containing 20 %, v/v, glycerol, with brucella broth,
and frozen at 270 uC, were subsequently homogenized with
PBS for microaerobic culture. Media used for culture
included trypticase soy agar with 5 % sheep blood (BAP),
medium impregnated with trimethoprim, vancomycin and
polymyxin (TVP; Remel), and medium impregnated with
cefoperazone, vancomycin and amphotericin B (CVA;
Remel). In addition, selective antibiotic medium (ABM)
contained the following components: blood agar base no. 2,
5 % horse blood (Remel), 50 mg amphotericin B ml21,
100 mg vancomycin ml21, 3?3 mg polymyxin B ml21, 200 mg
bacitracin ml21 and 10?7 mg nalidixic acid ml21 (Sigma). A
portion of each homogenized sample was applied directly to
TVP, CVA, and ABM media, and filtered through a 0?45 mm
pore-size filter and plated on BAP. Plates were incubated at
37 uC under microaerobic conditions for 2–4 weeks in
vented jars containing N2, H2 and CO2 (80 : 10 : 10).
Bacterial isolates with characteristic growth on agar and
microscopic morphology were subsequently used for
biochemical analysis and to obtain genomic DNA for
PCR, RFLP and 16S rRNA gene sequencing analyses.
Case report
An adult male baboon (Papio anubis) was euthanized due to
a chronic wasting disease. Prior to euthanasia with an
intravenous barbiturate, blood samples were collected for
haematological analyses, and values were compared to
clinical reference data for baboons (Hainsey et al., 1993).
The complete blood count was within the normal range;
however, the biochemical profile revealed hyperglycaemia
(192 mg dl21; normal range 65–101 mg dl21), hypertriglyceridaemia (315 mg dl21; normal range 36–94 mg dl21),
hyperlipasaemia (25 U l21; normal range 0–19 U l21)
and hypoproteinaemia (5?7 mg dl21; normal range
6?3–7?9 mg dl21). On gross examination during necropsy,
the liver appeared moderately enlarged. Representative
samples were collected from various organs, fixed in 10 %
neutral-buffered formalin, and processed for routine
histopathological evaluation.
Abbreviation: EHS, enterohepatic Helicobacter species.
46707 G 2006 SGM
Microaerobic bacteria were isolated from the ileum, liver
and colon. The isolates grew at 37 uC, at 42 uC, and in the
presence of 1 % glycine. They were oxidase positive, weak
catalase positive, urease negative, were unable to reduce
nitrate, and were negative for alkaline phosphatase hydrolysis, indoxyl acetate hydrolysis and gamma glutamyl
transpeptidase activity. The isolates from the ileum and
liver were resistant to nalidixic acid (30 mg disk) but
sensitive to cephalothin (30 mg), while the colon isolate was
sensitive to nalidixic acid and resistant to cephalothin.
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A. Garcı́a and others
99-5504) obtained from the colon of a non-diarrheic rhesus
monkey with chronic idiopathic colitis (Fox et al., 2001b).
The RFLP pattern of the colonic isolate was identical to the
pattern of an isolate of Helicobacter cinaedi (MIT 99-10781)
obtained from the faeces of a rhesus monkey (J. G. Fox and
others, unpublished results) (Fig. 1).
Fig. 1. PCR-RFLP patterns of the 16S rRNA gene of various
Helicobacter isolates from monkeys. (a) DNA digested with
AluI; (b) DNA digested with HhaI. Lanes: M, 1 kb plus ladder;
1, MIT 99-5504 (‘H. macacae’) (Fox et al., 2001b); 2, MIT 037674, ileum (baboon); 3, MIT 03-7674, liver (baboon); 4, MIT
00-6197 (H. cinaedi), liver (rhesus monkey) (Fox et al., 2001a);
5, 03-7674, colon (baboon); 6, 99-10781, faeces (rhesus
monkey).
PCR was performed on individual bacterial isolates using
Helicobacter genus-specific primers C97 and C05, as
previously described (Fox et al., 1998). These primers
were used to generate a 1200 bp PCR product of the 16S
rRNA gene from Helicobacter species that was then subjected
to RFLP (Fox et al., 1998). RFLP analysis revealed that ileal
and hepatic isolates had identical banding patterns with AluI
and HhaI restriction enzymes, but were distinct from the
banding patterns of the colonic isolate. The RFLP patterns
from the ileal and hepatic isolates were identical to
the pattern of an isolate of ‘Helicobacter macacae’ (MIT
In order to determine the phylogenetic relatedness of the
Helicobacter isolates, 16S rRNA sequencing and data
analyses were performed as previously described (Fox
et al., 2001b). The sequences of the 16S rRNA genes of
the isolates from the ileum and liver were 99 % similar to that
of ‘H. macacae’ (MIT 99-5501 and MIT 99-5504). The ileal
isolate had an intervening sequence (IVS) that was not
present in the liver isolate. The sequence of the 16S rRNA
gene of the colonic isolate was 99 % similar to that of the
Mainz strain (CCUG 33804) of H. cinaedi (Fig. 2).
Histological examination of the liver revealed that there was
multifocal and mild portal infiltration by low to moderate
numbers of lymphocytes and occasional macrophages
(Fig. 3a). The hepatocytes were diffusely and moderately
swollen with clear cytoplasm containing PAS-positive
material (glycogen) (Fig. 3b). Pancreatic islets of
Langerhans were diffusely effaced by deposits of pale
eosinophilic hyaline extracellular material that demonstrated apple-green birefringence under polarized light after
staining with Congo red (amyloid; Fig. 3c, d). In the caecum
and colon (ascending, transverse and descending), there was
mild to moderate separation of the crypts, and the lamina
propria was mildly expanded by low numbers of lymphocytes, occasional plasma cells, macrophages, rare globular
leukocytes and eosinophils (Fig. 3e, f). The inflammatory
infiltrates in the lamina propria of the descending colon
were slightly more prominent.
Fig. 2. Phylogenetic tree constructed on the basis of 16S rRNA sequence similarity values. The scale bar is equal to a 3 %
difference in nucleotide sequences as determined by measuring the lengths of the horizontal lines connecting two species.
GenBank accession numbers are given in parentheses. Isolates from this study are shown in bold type.
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Journal of Medical Microbiology 55
Enterohepatic Helicobacter species from a baboon
(a)
(b)
(c)
(d)
(e)
(f)
Discussion
This report describes the isolation of EHS from the ileum,
liver and colon of a baboon with pancreatic islet amyloidosis
and hepatitis. Although a previous study has reported the
histologic detection of Helicobacter-like organisms in the
stomach of baboons with gastritis (Mackie & O’Rourke,
2003), to the best of our knowledge this is the first report of
EHS being isolated from a baboon. ‘H. macacae’ was isolated
from the ileum and liver. ‘H. macacae’ is a novel species that
has been isolated from rhesus monkeys with and without
chronic idiopathic colitis (Fox et al., 2001b; J. G. Fox and
others, unpublished results). ‘H. macacae’ (MIT 99-5504)
has also been identified by PCR and sequence analysis in the
stools of children with a history of gastroenteritis and with
stool samples that were transiently positive for H. pylori
antigen by ELISA (Haggerty et al., 2005).
H. cinaedi was isolated from the inflamed colon of the
baboon. This strain was 99 % similar to the Mainz strain of
H. cinaedi. The Mainz strain was originally isolated from the
joint effusion of an AIDS patient with septic arthritis, and is
4 % distant from the H. cinaedi type strain (Dewhirst et al.,
2005; Husmann et al., 1994). In humans, H. cinaedi is the
most commonly reported EHS, and has been recovered
http://jmm.sgmjournals.org
Fig. 3. Representative sections of the liver
(a, b), pancreas (c, d), caecum (e) and
colon (f) from an adult male baboon. (a)
Multiple lymphoid aggregates in a large
portal triad. (b) Mild to moderate accumulation of magenta PAS-positive glycogen in
the hepatocytes. (c) Section of pancreas
with complete effacement of the islets of
Langerhans by pale eosinophilic hyaline
material (arrow) consistent with amyloid.
(d) Serial section of panel (c) stained with
Congo red and viewed under polarized light.
Note the apple-green birefringence of the
amyloid. (e) Section of the caecum with mild
to moderate separation of the crypts and
infiltration of the lamina propria by lymphocytes and macrophages. (f) Higher magnification of the colon depicting the proprial
infiltrates (arrow) comprised mostly of lymphocytes, occasional macrophages and eosinophils. Bars: (a, b), 100 mm; (c, d, f), 50 mm;
(e), 200 mm.
from patients with diarrhoea, bacteraemia and other
inflammatory conditions (Simons et al., 2004; Taylor et al.,
2003). In a survey ascertaining the prevalence of enteric
helicobacters using PCR-denaturing gradient gel electrophoresis (DGGE), in faecal samples from zoo animals, H.
cinaedi was detected in a captive baboon (Al-Soud et al.,
2003). H. cinaedi has also been isolated from the colon, liver
and mesenteric lymph node of a rhesus monkey with
chronic colitis and hepatitis (Fox et al., 2001a). H. cinaedi
frequently colonizes captive rhesus monkeys without overt
diarrhoea (Fernandez et al., 2002). Rhesus monkeys, in a
similar manner to hamsters as well as other animals, could
serve as potential reservoir hosts for human infection
(Fernandez et al., 2002; Gebhart et al., 1989; Vandamme
et al., 2000). Captive cotton-top tamarins (CTTs) with
endemic colitis are also colonized with a novel intestinal
Helicobacter species (Saunders et al., 1999). EHS have also
been identified in human patients with inflammatory bowel
disease (Bohr et al., 2004).
The accumulation of glycogen in the liver of the baboon, as
well as the lymphohistiocytic portal lesions, suggested
diabetes with hepatic involvement (Stone & Van Thiel,
1985). However, further clinical pathological evidence was
not available to support a definitive diagnosis of chronic
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A. Garcı́a and others
hyperglycaemia and diabetes. The hepatitis and typhlocolitis
were consistent with the lesions observed in rhesus monkeys
infected with EHS (Fox et al., 2001a,b). The islet amyloid is a
pathological feature in human type 2 diabetes that has also
been identified in baboons with and without hyperglycaemia, as well as in other species of non-human primates
(Hubbard et al., 2002; Kahn et al., 1999; Wagner et al., 2006).
Interestingly, nine of 40 (22?5 %) baboons with pancreatic
islet amyloidosis had concurrent diarrhoea (Hubbard et al.,
2002). In addition, the probable cause of death in 14 of 40
(35 %) baboons with pancreatic islet amyloidosis was
diarrhoea, colitis or typhlitis (Hubbard et al., 2002).
The occurrence of pancreatic islet amyloidosis in the baboon
may be unrelated to the infection with EHS. However, it is
interesting to note that in humans, the association between
H. pylori infection and type 2 diabetes remains controversial
(Dore et al., 2000; Gillum, 2004; Gulcelik et al., 2005; Ko
et al., 2001; Quadri et al., 2000). There is additional evidence
for the occurrence of pancreatic disease in the context of H.
pylori infection (Manes et al., 2003). Furthermore, in a study
of Syrian hamsters, isolation of Helicobacter cholecystus
correlated strongly with the presence of cholangiofibrosis
and centrilobular pancreatitis (Franklin et al., 1996). The
authors hypothesized that the pathological lesions in these
hamsters could have been the result of an ascending
infection of the common duct joining the bile and
pancreatic ducts (Franklin et al., 1996).
Amyloid deposits occur in association with several distinct
diseases and can be classified into two general forms,
systemic and localized (Hull et al., 2004). Secondary
amyloidosis is systemic and associated with chronic
inflammatory disease. On the other hand, in type 2 diabetes,
the amyloidosis is localized and islet amyloid polypeptide or
amylin is deposited in the pancreatic islets (Hull et al., 2004).
Amylin may be induced by oxidative stress, and once formed
may be responsible for beta cell death through the process of
oxidative stress (Hayden & Tyagi, 2002; Schubert et al.,
1995). Although we only observed inflammation in the
intestine and liver, and not in the pancreas of the baboon, it
is conceivable that increased oxidative damage produced as
a result of Helicobacter species infection may have directly or
indirectly affected the pancreas (Sipowicz et al., 1997).
Future experiments designed to investigate the pathogenic
role of EHS in metabolic diseases are warranted.
Bohr, U. R., Glasbrenner, B., Primus, A., Zagoura, A., Wex, T. &
Malfertheiner, P. (2004). Identification of enterohepatic Helicobacter
species in patients suffering from inflammatory bowel disease. J Clin
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Chen, T., Paster, B. J. & Fox, J. G. (2005). Discordant 16S and 23S
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Acknowledgements
This work was supported by R01CA67529, R01AI50952, R01DE10374,
T32RR07036, P01CA26731 and P30ES02109 from the National
Institutes of Health. The authors thank Robert P. Marini and Nirah
H. Shomer for their contribution to this study.
Haggerty, T. D., Perry, S., Sanchez, L., Perez-Perez, G. &
Parsonnet, J. (2005). Significance of transiently positive enzyme-
linked immunosorbent assay results in detection of Helicobacter
pylori in stool samples from children. J Clin Microbiol 43, 2220–2223.
Hainsey, B. M., Hubbard, G. B., Leland, M. M. & Brasky, K. M.
(1993). Clinical parameters of the normal baboons (Papio species)
and chimpanzees (Pan troglodytes). Lab Anim Sci 43, 236–243.
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