Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
Rectal experimental infection with H.pylori in
mice and rabbits
Qasim Najim Aubaid Al-Thewaini
Babylon University, College of Science, Biology Department\ Microbiology
Oruba Kuttof Hussein Al-Bermani
Babylon University, College of Science for women, Biology Department\
Microbiology
Abstract
Helicobacter pylori infection in the stomach is etiologically closely associated with chronic active
gastritis, peptic ulcer, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. In the
present study a clinical isolate of H. pylori obtained from dyspeptic patient, was used for experimental
infection. An experimental model for H.pylori infection has been established by rectally challenging
twenty one animals (mice or rabbits) with a different concentration of bacterial inoculums (1.5×109
,108, 107, 106, 105, 104, 103 CFU) for three consecutive days. Three animals were inoculated with
sterilised brucella broth as control group . All the animals were killed at 2 weeks postinoculation and
fragments of stomach, duodenum ileum, colon and rectum were collected and processed for culture and
histopathological examination. The present study shows that clinical isolate has been adapted to
colonize mice and rabbits. The minimum infectious dose of H.pylori required to colonize mouse
models was 106 CFU . while in rabbit models, the minimum infectious dose required to colonize
animals was 105 CFU. 1.5×109 CFU was selected to induce infection using rectal infection route in
nine experimental animals (mice or rabbits) and follow histopathological alterations at 2, 4, and 8
weeks postinoculation , in addition, bacterial reisolation was achieved at all time points. The result
shows infected mice with H.pylori exhibited chronic superficial gastritis. On the other hand infected
rabbits exhibited sever chronic gastritis with development of gastric ulcer at 8 weeks postinoculaton .
1: Introduction
Helicobacter pylori is a curved or spiral gram-negative polyflagellated bacteria which
can colonize the human stomach (Marshall and Warren, 1983; Kuster et al 2006;
Buso et al, 2009).
H. pylori is a human pathogen associated with type B gastritis, peptic ulcer disease,
gastric cancer and gastric Mucosa Associated Lymphoid Tissue (MALT) lymphoma.
As a result, the World Health Organization has classified H. pylori as a Class 1
carcinogen, (Wang et al, 1997; Raderer, 2000; Wu et al, 2001; Aguemon et al, 2004;
Eslick, 2006)
The primary disorder, which occurs after colonization with H. pylori, is
chronic active gastritis. This condition can be observed in all H. pylori-positive
subjects. The intragastric distribution and severity of this chronic inflammatory
process depends on a variety of factors, such as characteristics of the colonizing
strain, host genetics and immune response, diet, and the level of acid production. H.
pylori-induced ulcer disease, gastric cancer, and lymphoma are all complications of
this chronic inflammation; ulcer disease and gastric cancer in particular occur in those
individuals and at those sites with the most severe inflammation. (Sipponen et
al,1998; Stolte, 2002; Kuster et al, 2006).
Soon after Marshall and Warren had fulfilled Koch’s postulates for H. pylori
and acute gastritis via the infamous selfinfection experiment (Marshall et al, 1985;
Morris et al, 1991), it was apparent that an animal model was required to allow
elucidation of the mechanisms governing disease development and the pathogenic
properties of H. pylori, as well as for testing the effects of treatment and vaccination
on the pathogenesis of H. pylori infection . Oral challenges with H. pylori resulted in
infection in monkey (Solnick et al , 2004) , Mongolian gerbil (Mastumoto et al, 1997;
324
Tokieda et al, 1999; Ogura et al, 2000; and Hallsund et al ,2010), guinea pig (Shomer
et al,1998; de Jonge et al, 2004), dogs (Rossi et al ,2004), cats (Esteves et al , 2000),
mice (Marchetti et al 1995; Rabelo-Goncalves et al, 2005; Zhang et al , 2007 ) and
gonotobiotic piglets , that has been successfully used in H.pylori experimental
gastritis , peptic ulcer and gastric cancer ( Eaton et al, 1998), but is currently no
longer widely used .
The aims of the present study are to investigate the pathogenicity of H.pylori
in mice and rabbits , through an investigating the ability of a clinical H.pylori isolate
to colonize animal models (mice and rabbits) ,comparing the histopathological
changes occurring in mice and rabbits, after experimental infection with H.pylori and
investigating, if H.pylori associated with inflammatory bowel disease, through rectal
challenge with H.pylori
2: Materials and methods
2-1: Biopsy specimens collection
Between August and October 2009 , three gastric mucosa biopsy specimens were
collected from each of sixty consecutive dyspeptic patients (39 males and 21 females,
age range 18-61 years) undergoing endoscopy at digestive system ,Educational
Marjan Hospital.
2-2: Sample processing technique
The 180 fresh specimens were transported in brain heart infusion broth
supplemented with horse serum (Xia et al, 1993) and taken immediately to
microbiology laboratory under a strict aseptic condition.
Biopsy specimen was homogenized in 1ml normal saline by sterile lancet. This
homogenate was inoculated onto H.pylori selective medium. (Piccolomini et al, 1997;
Davood et al, 2009)
2-3: H. pylori isolation
The biopsy specimen homogenate was plated onto H.pylori selective medium
supplemented with horse serum. Test plate was incubated at 37C◦ for 5-7 days under
microaerophilic condition (Co2 generating kit (Oxoid) with manually pumping of
nitrogen gas. The clinical isolates were identified on bases of colony characteristics
such as nonhemolytic ,pinhead ,convex ,circular with (1-2 mm diameter ), colorless
and translucent colonies , microscopic characteristics of organism such as, spiral
shaped or curved rod bacteria, and biochemical characteristics like positively for
urease, catalase and oxidase tests, the resistance for Nalidixic acid (30 µg) and
sensitivity for Cephalothin(30 µg) additionally to other biochemichemical testes.
2-4: Bacterial inoculum preparation
Bacterial inoculum was prepared from a pure culture according to Shomer et al
(1998) and Rabelo-Goncalves et al (2005) methods, which includes the following
steps:
1- H.pylori was grown in brucella broth supplemented with horse serum at 37C◦ for
48 hours under microaaerophilic condition.
2- Broth culture was examined microscopically by modified Gram- staining for purity.
3- Bacteria were pelleted at 3500 r/m for 10 minutes
4- Bacterial pellet was resuspended in brucella broth .
5- Bcterial suspension was adjusted with Macfarland tube No 5 to a concentration of
approximately 1.5×109 CFU/ml .
From above bacterial inoculum, serial dilutions were prepared of 108, 107, 106, 105,
104 and 103 CFU/ml for determination the infectious dose of H.pylori
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Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
1.5×109 CFU H.pylori were selected to induce experimental infection in animal
models (Yan et al, 2004), then clinical outcomes of H.pyori infection were followed
at 2, 4, and 8 weeks postionculation
2-5: Experimental H. pylori infection
2-5-1: Experimental animals
I- Mouse model
Thirty three female BALB/c mice were 7-8 weeks old, when challenged. Body weight
was 30-32g. They were obtained from animal house, College of science, Babylon
University. The mice were housed in an air conditioned room with 12 hour light, 12
hour dark cycle. They were fed a commercial rodent diet with free access to drinking
water.
II- Rabbit model
Thirty three rabbits here were from established inbred lines and were purchased
from animal house, College of Medicine, Kufa University. These rabbits were
designated as a young adult, they were 6-8 month old with body mass of 1.5-1.75 kg.
The rabbits were fed a commercial chow, or grasses, with free access to drinking
water
2-5-2:Experimental design for determination the infectious dose of H. pylori
twenty one animals consisted of three animals per group with no prior H.pylori
infection were each inoculated rectally on a single occasion with 109, 108, 107, 106,
105, 104 or 103 CFU of bacteria. In mouse models, mice were fasted for 24 hour, Half
an hour later, each three mice challenged with 0.5ml of one type of bacterial inoculum
concentrations, for three consecutive days (Wang et al, 1997; Ayraud et al, 2002) .
Three control mice were inoculated with 0.5ml sterilized brucella broth.
In rabbits model, they were fasted for 24 hour. Half an hour later, 3ml of each
concentration of bacterial inoculums was given to each group, which consisted of
three rabbits, for three consecutive days. Three control rabbits were received orally
3ml brucell broth.
2-5-3: Experimental design for induction gastritis using 1.5×109 CFU
Nine animals (mice or rabbits) with no prior H.pylori infection were challenged
rectally with 109 CFU of H.pylori. Mice fasted for 24 hour , were inoculated with
0.5ml of bacterial inoculum. The rabbits fasted for 24 hour, and were administreated
with 3ml of the bacterial inoculum.
3: Results
3-1: mouse model
3-1-1: Infectious dose of H.pylori in infected mice
At 2 weeks postinoculation ,cultures of biopsy specimens from the gastric antrum of
inoculated mice with 103 and 104 and 105 CFU were negative , whereas one of three
animals of each group that inoculated with 106 ,107 , CFU were positive culture . Two
of three animals that inoculated with 108 and 109 were positive culture, (Table-1).
Histopathological examination of gastric antrum showed , that, inoculum 104 CFU or
more was able to induce mild gastritis in rectally challenging mice ,while animals that
inoculated with 103 did not exhibit any histopathological changes in gastrointestinal
tract. H.pylori was not recovered from duodenum, ileum, colon and rectum of the
infected animals. Additionally the histopathological changes were not observed in
these parts of the alimentary tract. Histological detection of bacteria in Giemsastained gastric section was positive in mice inoculated with 106 or more bacteria
326
Table-1: Inoculum concentration of H.pylori and gastric colonization of the infected
mice
Inoculum (CFU)
(mice No is 3)
infected mice
9
10
3
2
108
3
2
7
10
3
1
6
10
3
1
105
3
0
4
10
3
0
103
3
0
Total
21
6
3-1-2: Experimental infection in mice using 1.5×109 CFU
I: Macroscopic finding
Mice did not exhibit clinical signs of gastritis. Decrease in weight of infected
mice was not noted at 2,4 and 8 weeks postinoculation. Additionally no visible
gasterointestinal alteration or lesion at necropsy in any of the mice .
II:Bacteriological evaluation
Biopsy specimens were obtained aseptically from the gastric antrum and corpus,
duodenum, ileum, colon and rectum were cultured separately at 2, 4 and 8 weeks
postioculation (Table -2). At 2 and 4 weeks postioculation gastric antrum biopsies
from two of three inoculated mice for each groups were culture positive . At 8 weeks
postioculation one of three animals were culture positive .While the intestine and
corpus biopsies that obtained from inoculated mice at all time points were culture
negative for H.pylori. H.pylori was observed in a Giemas –stained sections of gastric
tissues
Table-2: Reisolation of H. pylori from infected mice
Infection time (wks)
No of mice
Positive culture
After dosing
Number-%
2
3
2:3(66.6)
4
3
2:3(66.6)
8
3
1:3(33.3)
Total
9
5:9(55.5)
III: Histopathology
In Rectally challenging animals with H.pylori ,there was histological evidence of
chronic superficial gastritis with infiltration of lymphocytes in gastric antrum .The
infiltration of inflammatory cells was seen at 2,4 and 8 weeks postinoculation . At 2
and 8 weeks postinoculation, the infiltration was mild while at 4 weeks post
inoculation it was moderate
and was present in lamina properia (Figure 1).
Histopathological changes in duodenum, ileum, colon and rectum were not observed
at all time points.
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Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
A
B
A
Figure-1: Normal gastric mucosa of the control mice, without inflammation (A);Chronic superficial
gastritis: Moderate chronic inflammatory cells infiltration in lamina properia challenged mice with
H.pylori, at 4 weeks postinoculation (B) .H&E.X400(B)
3-2-Rabbit model
3-2-1: Infectious dose of H.pylori in infected rabbits
Infectious dose of H.pylori in rectally challenging rabbits was estimated
.H.pylori was recovered from animals that received 105 CFU or more bacteria , while
it was not recovered from animals that inoculated with 103 and 104 . (table-3).One of
three animals inoculated with 105 ,106 and 107 and 108 were culture positive.two of
three animals inoculated with 109 was positive culture .Histopathological analysis was
found that animals inoculated with 103 and 104 developed mild chronic gastritis
,while animals received 105 CFU or more bacteria developed moderate chronic
gastritis. The duodenum, ileum, colon and rectum were negative for both H.pylori
culture and for histopathological changes. By using Giemas stain technique, H.pylori
was observed in animals inoculated with 106 CFU or more bacteria
Table-3: Inoculum concentration of H.pylori and gastric colonization in infected
rabbits
Inoculum (CFU)
Rabbit No (3)
Infected rabbit
109
108
107
106
105
104
103
Total
3
3
3
3
3
3
3
21
2
1
1
1
1
0
0
6
3-2-2:Experimental infection in rabbits using 1.5× 109 CFU
I:Macroscopic finding
Decrease in weight and loss of appetite of inoculated animals were noted at all
time points, stomach enlargement was also noted but without visible gastric lesion at
necropsy in any inoculated animals
II:Bacteriological evaluation
Gastrointestinal tract colonization by H. pylori was assessed in the present study.
Mucosal biopsy specimens were obtained for bacteriological evaluation from
stomach, duodenum, ileum, colon and rectum of the necropised animals at 2, 4 and 8
weeks postinoculation
(Table-4). H.pylori was recovered from gastric antrum,
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whereas duodenum, ileum, colon and rectum gave negative result for H.pylori culture.
At 2 weeks, two of three animals were culture positive, while at 4 and 8 weeks
postinoculation one of the three animals of each groups was culture positive ,whereas
rectum biopsies gave negative results for H. pylori culture
Table-4: Reisolation of H.pylori from infected rabbits
Infection time (wks)
No of rabbits
After dosing
2
3
4
3
8
3
Total
9
Positive culture
No-%
2:3(66.6)
1:3(33.3)
1:3(33.3)
4:9(44.4)
III: Histopathology
The histopathological examination of the gastrointestinal tract of rectally
challenging rabbits with H. pylori was achieved at 2,4,and 8 weeks postinoculation
.At 2 weeks postinoculation ,histological section of gastric antrum explained
moderate chronic gastritis ,while at 4 weeks postinoculation, animals developed
chronic superficial gastritis, with sever chronic inflammatory cells infiltration and
degeneration of the mucosal epithelial lining cells , (Figure-2) . At 8weeks
postinoculation ,ulceration ,with moderate chronic inflammatory cells infiltration
were seen in the gastric antrum ,(Figure-10). Infiltration of inflammatory cells was
lymphocytic and was present in the lamina propria. Histological examination of the
duodenum, ileum, colon and rectum at 2, 4 and 8 weeks did not show any
pathological changes.
B
A
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Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
Figure-2: Histological sections of gastric antrum of rectally challenged rabbit with H.pylorichronic
superficial gastritis, sever chronic inflammatory cells infiltration in the lamina properia at 4 weeks
postinoculation (A) H&E.X400; gastric ulcer at 8 weeks postinoculation (B), H&E.X100
4:Discussion
4-1; Mouse model
4-1-1: Infectious dose of H.pylori in infected mice
The rectal challenge with H.pylori as an experimental infection route ,was
attempt to shed light on the mechanisms whereby H.pylori is acquired and causes
disease . The infectious dose in rectally challenged mice was determined ,which was
106 CFU, it depended on the cultural finding of the gastric biopsy specimens .This
infectious dose was higher than those recordedby other worker, when using oral
experimental infection. This discrepancy may be associated with many reasons ,such
as defecation process ,which makes clearance of organism ,so diluted bacterial
inoculum may reach stomach which can not colonize stomach or induce gastritis
.Another reason may be that ,H.pylori is a fragile bacteria and does not survive in an
alkaline environment (Mullines and Steer ,1998) .
Therefore bacterial cells may die when inoculated by inverse route when passed
throughout intestine to stomach, in addition to resident intestinal bacterial flora that
competes H .pylori and suppresses colonization (Shomers et al, 1998).This cultural
finding was in conflict with those of histopathological finding ,it showed that all
animals inoculated with 104 CFU or more bacteria exhibited mild gastritis, this may be
due to the presence of H.pylori in the mouse stomach at undetectable level by culture
,or may be a result of the existence coccoid form of bacteria in stomach ,this form is
unculturable in vitro (Wang et al , 1997) . Bacterial detection by Giemsa –stained
histological sections , showed that ,all animals inoculated with 106 or more bacteria
were positive for H.pylori . Asimilar finding was reported by Van Doorn et al (1999) .
4-1-2: Experimental H.pylori infection in mice using 1.5× 109 CFU
The rectal challenge with H.pylori was one of the proposed experimental infection
route of mice to know whether H.pylori was associated with Inflammatory Bowel
Disease (IBD). The present study has evidence for that H.pylori did not colonize
rectum or colon ,it colonized only stomachs of inoculated mice ,as indicated by the
cultural finding of the stomach and large intestine biopsy specimens. H.hepaticus was
first discovered in association with hepatitis ,hepatocellular adenoma and
adinocarcinoma in some strains of mice . However the primary site of colonization is
the cecum and colon in mice that develop liver lesion or not develop liver lesion
(Foltz et al 2004 ) . In some immune deficient strain of mice ,H.hepaticus has been
associated with colitis and proctitis characteristic of IBD . Histopathological
examination explained that all animals exhibited mild gastritis like those orally
inoculating mice.
4-2:Rabbit model
4-2-1: Infectious dose of H.pylori in infected rabbit
Rectal challenge with H.pylori was a successful experimental infection route to
colonize animals and to induce gastritis .The minimum infectious dose of H.pylori
required to colonize animals was 105 CFU , like those recorded of orally challenging
rabbits .
The infectious dose of H.pylori remains unknown as data from three volunteer
experiments are inconclusive .Marshall et al (1985) , achieved a successful infection
with clinical isolated strain at a dose of 109 CFU in a small liquid feed following the
330
use of antacid . A second volunteers failed to became infected with a dose 4×107 CFU
of the same strain (Morris et al , 1987) , infection did succeed in the second
volunteers when the antacid was used with different strain at a dose of 3×105 CFU
.(Morris et al , 1991) .However these experimental doses may be excess of the
number of organism required to cause natural infection (Young et al ,2000).
Histopathological examination showed that animals inoculated with 103 or 104 CFU
exhibited mild gastritis while animals inoculated with 105 CFU or more bacteria
developed moderate gastritis, these pathologic change were similar to those observed
in orally challenging rabbits. Giemsa –stained histological section was used to
evaluate colonization of organism in the gastric mucosa, it showed, there were
positive results of animals inoculated with 106 CFU or more bacteria, similar finding
was reported by lee et al (1997).
4-2-2: Experimental H.pylori infection in rabbits using 1.5× 109 CFU
Rectal challenge with H.pylori had been documented to colonize animals and result
gastritis .Macroscopic findings such as ,vomiting , and loss of appetite were also
noted .These findings were consistent with those recorded by Ikeno et al (1999).
During the course of the experimental infection ,the gastric biopsies from infected
animals were cultured and gave positive results for H.pylori . These findings were
coincided with those reported by Fox et al (1995) ,Shomer et al (1998) and Koga et al
(2003 ). Koga et al (2003), found that in the experimental infection of miniature big
,the bacterial colonization increased gradually , suggested that the cell receptor of
organism may be increased as a consequence of ulcer formation .
H.pylori was not isolated from large intestine ,other instances ,these regions were
not colonized by H.pylori .However ,Buso et al (2009) , found that a greater
prevalence of colon adenomas in patients infected with H.pylori. H.hepaticus
colonization persisted in the large intestine and liver ,it was associated with
inflammatory bowel disease in nude and SCID mice ,manifested as rectal prolapse
accompanied by colitis and proctitis (Whary and Fox , 2004). H.trogontum and
H.bilis were isolated from the colon of naturally infected Wster and Holtzman rats
(Mackie and O,Rourke , 2003). Histopathological examination showed that the
animals developed sever chronic gastritis with superficial erosion on the surface of
gastric mucosa and gastric ulcer .These pathologic changes were similar to those
observed in H.pylori- infected persons .Gastric ulcer were noted in miniature pig
infected with H.pyloti (Koga et al 2003 ), Mongolian gerbils (Ikeno et al ,1999;Kuster
et al 2006 ;and Hallersund et al ,2010 ).
Acknowledgement
Thanks are due to the staff in teaching Marjan Babylon Hospital especially
those working in the digestive system unit , and to Dr. Akeel Al-Mhel for his help in
obtaining biopsy specimens. Acknowledgments go to Professor . Asa,ad Abed AlHamza Al Janabi (Kufa University) and to Dr. Mazin Ja,afar Mussa (Karblaa
University), for their assistance in histopathology.
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