Study of the preventive effect of anti-urease IgY on helicobater pylori infection in white mice

The aim of this study is to evaluat the inhibitory effect of IgY against H. pylori-derived urease on H. pylori in experimental animals.

STUDY OF THE PREVENTIVE EFFECT OF ANTI-UREASE IGY

ON HELICOBACTER PYLORI INFECTION IN WHITE MICE

Do Hoang Long*; Do Minh Trung**; Vi Thuat Thang***; Tran Ngoc Dung***

Le Thu Hong***; Le Van Dong**; Nguyen Anh Tuan**

SUMMARY

Objectives: Urease inhibition with oral specific antibody is a new approach in prevention and

treatment of Helicobacter pylori infection This research aimed to investigate the preventive

effect of anti-urease IgY on H pylori infection in experimental animal Subjects and method:

White mice were prophylactic treated by having food with or without anti-urease IgY a week before H pylori inoculation and continuously till end of the monitoring time Nine weeks after the start of the diet, the mice were examined to determine the reactivity of anti-H pylori IgG in

serum and the inflammation and infection status of the stomach biopsy Results: There were

significant differences in the anti-H pylori IgG titer, the eradication of H pylori and the reduction

of H pylori-associated gastritis between the two groups of infected mice Conclusion: Oral

administration of urease-specific IgY prevented H pylori colonization in the inocculated mice

* Keywords: Helicobacter pylori; Anti-urease IgY; Oral passive immunization; Preventive effect

INTRODUCTION

In Vietnam as well as in the world,

Helicobacter pylori (H pylori) infection

accounts for 75 - 95% of all causes of

gastritis and peptic ulcer disease, in which

urease secreted by the bacteria is essential

for the survival of the bacteria, facilitating

the bacteria's invasion into the gastric

mucosa [4] In spite of new antibiotics to

be used recently, failure in treatment of

H pylori induced gastritis and duodenal

ulcer is common, due to the antibiotic

resistance of the bacteria, especially in the

developing and least developed countries

[5, 7] Since then, prophylactic treatment

with drug or food containing compound

that inhibite the bacteria before they get cololization become a new and promissing approach in developed contries such as Korea and Japan [6]

Similar to mammals, hens are capable

of creating antibodies against pathogen microbes; these antibodies can be transferred from a hen's blood into the egg yolk laid

by the hen itself, hence its name IgY (yolk immunoglobulin) Technology and procedures for antigen-specific IgY production have been established elsewhere [3] IgY has been used orally as a passive immunization method in prevention and treatment of some diarrheal diseases of animal and human

* Cantho Universiry

* Vietnam Military Medical University

** 103 Military Hospital

Corresponding author: Le Van Dong (levandong@yahoo.com)

Date received: 10/10/2017

Date accepted: 21/11/2017

IgY against urease of H pylori might be

effective in preventing H pylori infection,

and/or reduce damages caused by the

bacteria on gastric mucosa This study

aims at: Evaluating the inhibitory effect

of IgY against H pylori-derived urease on

H pylori in experimental animals

SUBJECTS AND METHODS

1 Subjects

- Experimental animal: 28 male Swiss

mice, weight 18 - 20 gr provided by the

Animal House, Department of Equipment

and Technical Materials, Vietnam Military

Medical University

- Other materials:

- H pylori (isolates HP3, HP16 and HP18)

from patient's gastric mucosa biopsy samples

at 103 Military Hospital in our previous

study [1]

- IgY against urease derived from the

3 isolates of H pylori (HP13, HP16,

HP18), purified at Department of

Immunology and Applied

Pharmaco-Medical Research Center, Vietnam Military

Medical University in our previous study [2]

2 Methods

* Preparing mice diet containing IgY

against H pylori's urease:

The food was prepared by mixing the

yolk protein suspension rich in IgY against

urease with conventional nutrition-enriched

mice food (4 mg IgY per 1 gr of food,

according to concentration estimation method

described by Nomura)

* Grouping of mice and experiments:

Mice were divided into 4 groups: group

1 and 2 (5 mice per group): non-H pylori

infection groups, of which mice in group 2

only were feeded with diet containing IgY against urease; group 3 (control group)

and group 4 (study group): H pylori

infection groups (9 mice per each group),

of which mice in group 4 only were feeded with diet containing IgY against urease

At day 7th, mice of group 4 were kept overnight without feeding, then infected in

the next morning with H pylori by esophageal

intubation (108 CFU/mL; 15 mL/kg body weight) After 9 weeks, blood samples of all mice were taken for determination of

anti-H pylori antibody titer; mice were all

sacrificed and the stomach of the mice were sent out for pathohistological analysis, with focus on severity of gastritis

and the presence of H pylori

RESULTS AND DISCUSSION

1 Animal activities during experiment

Within first 9 weeks of experiment, all mice of the groups were in normal activities, except mice in groups 3 and 4 showing less activities, refusal of eating, and ruffled hair Mice in group 3 showed weight loss after infection and during the following weeks until experiment ended

2 Effect of diet containing IgY against urease on body weight of the mice

In non-H pylori infected mice, average

body weight (BW) of the mice in group 1 and 2 increased during the experiment, being 16.8 and 16.4 grs at the beginning

of the experiment, respectively, and 18.2 grs

at the end of the experiment There was

no significant difference between BW of the mice in the 2 groups In addition, diet containing IgY against urease had no significant effect on mice’s body weight

Conversely, in H pylori infected mice,

average BW of the mice in group 3

decreased, being 17.5 grs at the beginning

of the experiment, and 17.4 grs at the end

of experiment (with net weight loss of

0.1 grs) Meanwhile, average BW of mice

in group 4 (having IgY against H

pylori-derived urease) were 17.3 grs and 19.1 grs

at the beginning and the end of experiment,

respectively (with net weight gain of 1.7 grs)

No significant diffenrence in BW of mice in

groups 3 and 4 was observed; however,

the weight gain of mice in group 4 revealed

a positive effect of IgY against H

pylori-derived urease (used as a preventive

measure) on development of H

pylori-infected mice

It was showed by our observation in

this study that, those mice feeded with

food containing IgY against H

pylori-derived urease (both in control group and

in H pylori-infected group) all had better

weight gain compared to the others This weight gain was probably due to the fact that IgY, by having protein’s nature, had provided more nutrition which helped mice developed better Interestingly, IgY

is a protein originated from hen’s egg, and

in this study, it showed no immunogenicity when orally administered to mice Taking all into account, these findings partly proved the non-toxic property of the IgY preparation, and thus, the safety of the preparation for oral administration Additionally, the increase in body weight

of H pylori-infected mice feeded with the

IgY preparation revealed the inhibitory

effect of IgY against H pylori-derived

urease on the bacteria

Graph 1 & 2: Average body weight of mice:

(1) non-H pylori infection, and (2) H pylori infection

3 Determination of anti-H pylori antibody in H pylori-infected mice

All blood samples from mice in group 3 (no prevention by anti H pylori-derived urease IgY) showed reactive with anti-H pylori antibody; meanwhile, only 66.6% of

samples from mice in group 4 (IgY prevention group) were reactive with the antibody

Additionally, activity of anti-H pylori antibody in H pylori-infected mice with IgY

prevention was lower than that of mice without IgY prevention (p = 0.003)

(2) (1)

4 Severity of gastric mucosa inflammation in mice

All H pylori-infected mice without anti-urease IgY prevention developed gastritis;

meanwhile, only 77.7% of mice infected with H pylori but had IgY prevention presented

with gastritis Besides, the gastritis situation in mice without IgY prevention was more

severe than that in mice with IgY prevention (p = 0.004)

5 Gastric submucosa’s bacterial density analysis

No presence of H pylori was recorded in gastric submucosa’s tissue samples of

mice in groups 1 and 2; meanwhile, the bacteria was present in all samples from

H pylori-infected mice without IgY prevention (group 3) Bacterial density recorded in

gastric submucosa’s tissue samples from mice in group 4 (IgY prevention group) was

lower (44%) Similarly, H pylori bacterial density on gastric mucosa of mice without IgY

prevention was higher than that of mice with IgY prevention (p  0.0001)

Graph 3 & 4: Gastritis in H pylori-infected mice: (3) severity of gastritis,

and (4) H pylori bacterial density

Figure 1 & 2: Histological gastric injuries in mice N3-C1: (1) H&E staining,

and (2) Giemsa staining

(3)

(4)

Regarding the preventive effect of IgY

against H pylori-derived urease, our findings

were in accordance with results published

by Nomura et al Firstly, all mice in group 4

in our study showed a reduced activity of

anti-H pylori antibody, yet a complete

eradication of the bacteria from gastric

mucosa in the H pylori-infected mice was

not fully observed Secondly, 55.6% of

mice in group 4 got a complete bacterial

eradication (HP testing negative), which

was lower compared to that in Nomura’s

study (100%); nonetheless, among HP (-)

mice in our study, 40% of mice was free

of gastritis, partly revealing that IgY against

H pylori-derived urease helped eradicate

the bacteria It was much likely that the

preventive effect of the IgY preparation

helped protecting the mice’s gastric mucosa

from the infection of H pylori; in other

words, the IgY worked by eradicating the

cause of gastritis The preventive effect of

IgY against H pylori-derived urease was

revealed by significant reduction of H pylori

bacterial density on the gastric mucosa

(p < 0.0001) Furthermore, the severity

of gastritis in mice of group 4 was

proportionally decreased along with the

decrease of H pylori on gastric mucosa

(p = 0.004)

In general, the H pylori infection rate

of mice in group 3 was the highest

compared to the others; in this group, the

activity of anti-H pylori antibody and the

severity of gastritis was higher than those

in the others as well This observation

suggested that IgY against H pylori-derived

urease had probably contributed to

improvement of H pylori-induced gastritis,

as well as decrease of urease activity at the gastric mucosa by inhibiting H.pylori’s

urease, and thus, eradicating the survival

factor for H pylori, leading to a reduction

of gastric mucosa’s injuries and making the bacteria itself easier to be eradicated

IgY against H pylori-derived urease

had been administered to the experimental

mice before H pylori infection, with the

aims of evaluating the preventive effect of the IgY preparation It was revealed by the results that in mice with IgY prevention, the infection rate and the gastritis severity were lower compared to those without IgY prevention The IgY preparation was proved to be non-toxic and safe by oral administration In other words, IgY against

H pylori-derived urease exerted a preventive

effect on H pylori infection

CONCLUSIONS

We have successfully established an

animal model of H pylori infection on Swiss mice by injecting H pylori suspension,

108 CFU/mL/mouse through esophageal intubation The infection rate was 100%

by anti-H pylori antibody testing on blood

samples and the presence of the bacteria

at the gastric submucosal tissue of mice being infected

A continuous diet containing IgY against urease (4 mg IgY/gram of food) for

1 week before infection and thereafter

showed a preventive effect of H pylori

infection on experimental mice

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