Study on protective effect of specific igy antibody on cholera toxin intoxicated suckling mice

Journal of military pharmaco-medicine no1-2018

STUDY ON PROTECTIVE EFFECT OF SPECIFIC IgY ANTIBODY
ON CHOLERA TOXIN-INTOXICATED SUCKLING MICE
Hoang Trung Kien*; Nguyen Anh Tuan*
Le Thu Hong**; Nguyen Dang Dung*
SUMMARY
Objectives: To evaluate the protective effect of anti-choleratoxin IgY antibody on
choleratoxin-intoxicated animal. Subjects and methods: Suckling mice (3 - 5 days old) were
intoxicated with choleratoxin, then treated with anti-choleratoxin IgY. The protective effect of IgY
antibody was evaluated by measuring survival time of choleratoxin-intoxicated suckling mice
treated with anti-choleratoxin IgY and by histopathological analysis of epithelial cells of the
mice's intestinal mucosa. Results: After 66 hours of choleratoxin infection, 65% of mice treated
with anti-choleratoxin IgY were still alive (compared to 0% in control group); pathohistological
images of the mice's intestinal mucosa showed less damages in those from mice treated with
anti-choleratoxin IgY antibody compared to controls. Conclusions: IgY antibody against cholera
toxin helps protect suckling mice from toxic effect of choleratoxin.
* Keywords: Vibrio cholerae; IgY antibody; Sucking mice.

INTRODUCTION
Cholera is a serious diarrhea disease
caused by gastrointestinal infection of Vibrio
cholerae. In 2016, 172,454 cholera cases
were reported by WHO, including 1,304
cases of deaths in 42 countries in the
world with fatality rate of 0.8%, and the
total number of cholera cases and deaths
has not decreased in the last five years,
but the fatality rate for cholera is till high.
Following the gastrointestinal infection,
the bacteria secretes choleratoxin (CT),

which consists of one A (active) and five
B (binding) subunits. The B subunits of
CT (CTB) can bind to GM1 ganglioside

expressing on epithelial cells of the
intestinal mucosa. Once bound, the
cleavage between subunit A1 and A2
segments is facilitated and A1 portion
moves into the cells. The A1 component
stimulates the production of adenylate
cyclase, leading to increased production
of cyclic AMP inside the cells. This
increased intracellular level of cyclic AMP
results in a disruption of the active
transport of electrolyte across the cell
membrane, which leads to fluid secretion
into small intestine. The diarrhea occurs
when the volume of fluid entering the
colon from intestine is over the reabsorptive capability of the colon.

* Vietnam Military Medical University
** 103 Military Hospital
Corresponding author: Nguyen Dang Dung ()
Date received: 20/10/2017
Date accepted: 18/12/2017

151


Journal of military pharmaco-medicine no1-2018

The utilization of antibodies specific to
V. cholerae exerted preventive and
therapeutic effects against cholera on
animal orally infected with live
V. cholerae. However, whether IgY
antibody specific to CT can protect animal
from cholera-like disease caused by oral
CT infection has not yet been completely
elucidated.
In this study, we developed an animal
model of CT infection and evaluate the
protective effect of specific IgY antibody
on CT-intoxicated suckling mice.

SUBJECTS AND METHODS
1. Subjects.
- Suckling mice (3 - 5 days old),
regardless of gender, provided by the
Animal House of Military Medical University.
- C0852 CT freeze-dried powder provided
by Sigma.
- IgY isolated from egg yolk laid of hens
immunized with CT by method described
previously [1, 3].
- Reagents used for immunohistochemical
(IHC) staining.

2. Methods.
* Establishing choleratoxin-intoxicated animal model:
Table1: Choleratoxin intoxication model on suckling mice.

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

(n = 5)

(n = 5)

(n = 5)

(n = 5)

(n = 5)

(n = 5)

Mice given

Mice given

Mice given

Mice given

Mice given with

Mice given with

with 50 µL of

with 50 µL of

with 50 µL of

with 50 µL of

50 µL of

50 µL of

choleratoxin

choleratoxin

choleratoxin

choleratoxin

choleratoxin

0.008 mg/mL

0.04 mg/mL

0.2 mg/mL

1 mg/mL

5 mg/mL

NaCl 0.9%

The experimental animal was suckling mice (3 - 5 days old), separated from their
mother 3 hours before the start of experiment. A total of 30 mice were devided into
6 groups, with 5 mice in each group.
In group 1 (control group): mice were given with 50 µL of NaCl 0.9% through
esophageal intubation using a plastic flexible needle; in the remaining groups (from
group 2 to 6), mice were given with 50 µL of CT at concentrations of 0.008; 0,04; 0,2;
1 and 5 mg/mL in NaCl 0.9%, respectively.
Thereafter, all the mice were inspected every 2 hours for diarrhea and death, of
which number of mice died in each group was recorded at 2-hour time intervals until all mice
in the CT-intoxicated groups died.
152


Journal of military pharmaco-medicine no1-2018
* Investigation of the protective effect of specific IgY antibody against CT on CTintoxicated suckling mice:
Table 2:
Group
Intervention
Choleratoxin intoxication
(0 hour)

Treatment
(3 hours after
choleratoxin intoxication)

1

2

3

4

(n = 20)

(n = 20)

(n = 20)

(n = 20)

50 µL
NaCl 0.9%

50 µL choleratoxin
1 mg/mL

50 µL choleratoxin
1 mg/mL

50 µL choleratoxin

1 mg/mL

50 µL
NaCl 0.9%

50 µL
NaCl 0.9%

50 µL

50 µL

IgY against Freund
adjuvant

IgY against
choleratoxin

4 groups of suckling mice (3 - 5 days
old), with 20 mice in each group, were
adopted for this experiment. Suckling
mice were separated from their mother
3 hours before testing.

number of mice died in each group
recorded at 2-hour time intervals until all
mice in group 2 (treated with NaCl 0.9%,
served as negative control-1 group) died.

In group 1 (biological control group):

mice were given with 50 µL of NaCl 0.9%
through esophageal intubation; in the
groups 2, 3, and 4, mice were given with
50 µL of CT at 1 mg/mL. Three hours
after CT injection, mice in the groups 1
and 2 were given with 50 µL of NaCl
0.9%; mice in group 3 were administered
with 50 µL of IgY isolated from egg yolk
laid of hens immunized with Freund
adjuvant only (served as negative control2 group), meanwhile mice in group 4 were
administered with 50 µL of IgY antibody
isolated from egg yolk laid of hens
immunized with a mixture of CT antigen
and Freund adjuvant.

Immunohistochemistry (IHC) technique

All the mice were then inspected every
2 hours for diarrhea and death, with

* Pathohistological analysis:
was adopted to determine CT on
intestinal mucosal epithelium, and to
evaluate the intestinal epithelial injuries of
the experimental CT-intoxicated suckling
mice.
Briefly, a mouse with anti-CT IgG antibody
(the primary antibody) was incubated with
intestinal specimen's slide. After being
washed, the slide was incubated with the

secondary antibody (a biotynated rabbitanti-mouse IgG antibody). The slide was
washed, and then incubated with an
avidin-peroxidase

conjugate.

Finally,

diaminobenzidine substrate was added
for slide's microscopic visualization and
analysis.
153


Journal of military pharmaco-medicine no1-2018
RESULTS AND DISCUSSION
1. Establishment of choleratoxin intoxication model on suckling mice.

Graph 1: Survival time of mice after choleratoxin intoxication.
In an attemp to establish an animal
model for CT infection, we have chosen
suckling mice, since the animal was
previously reported to be susceptible to
cholera caused by gastrointestinal infection
with live V. cholerae. In the present study,
we investigated the susceptibility of the
mice with 5 different doses of CT infection.
Mice in group 1 served as biological control
group, all of which were given NaCl 0.9%
only, to ensure that an the esophageal

intubation and injection of the solutions
did not unexpectedly kill the mice.
Graph 1 presented the results of
establishment of the CT-infection animal
model, with a single injection (through
esophageal intubation) of 50 µL of CT at
0.008 mg/mL; 0.04 mg/mL; 0.2 mg/mL;
1 mg/mL; 5 mg/mL for each suckling
154

mice, respectively. CT infection could only
kill the experimental animals significantly
when using a CT solution at concentration of
1 mg/mL and 5 mg/mL (group 5 and
group 6), with all mice died at 58 and 66 hours
following CT intubation, respectively. No
significant difference in survival rate and
time of CT-intoxicated mice in the two groups
(intoxicated with 1 mg/mL and 5 mg/mL of
CT, respectively) was observed.
The survival rate of mice in group 1
(non-CT infection group) and in groups 2,
3 and 4 (intoxicated with CT at
concentrations of 0.008, 0.04 and
0.2 mg/mL, respectively) at 66 hours
following CT infection ranged from 60 to
80%. The survival time of mice in group 1
compared to those in groups 5 and 6 was
significantly different (p < 0.01).

Journal of military pharmaco-medicine no1-2018
Analysis on pathohistological images
of intestinal mucosa from CT-intoxicated
mice revealed obvious presence of
choleratoxin on intestinal mucosa (figure 2B),
and epithelial cells' damages (figure 2A)
were not observed in specimens from

(A)

non-CT-intoxicated mice (figure 1A) which
showed negative choleratoxin staining by
immuno-histochemical staining method
(figure 1B); this is in accordance with the
role of CT in pathogenesis of V. cholerae
infection.

(B)

Figure 1: Normal intestinal mucosa of mice; (A): HE staining, X400;
(B): immuno-histochemical staining, using anti-choleratoxin antibody,
X400: choleratoxin (-).

(A)

(B)

Figure 2: Intestinal mucosa of mice intoxicated with CT: (A) CT-intoxicated, no anti-CT
IgY treatment; HE staining, X400; (B) CT-intoxicated, no anti-CT IgY treatment;

immuno-histochemical staining with anti-choleratoxin antibody, 400X: choleratoxin (+).
It was therefore revealed by the results that oral CT intubation can cause death to suckling
mice (3 - 5 days old), and the death was likely due to CT-induced cholera-like disease.
155


Journal of military pharmaco-medicine no1-2018
2. Protective effect of anti-choleratoxin
IgY on choleratoxin-intoxicated mice.

likely due to the specificity of the IgY to
CT; in other words, it was very likely that
the CT-specific IgY bound to the CT
molecule and therefore neutralized it, thus
exerting the therapeutic effect as observed.

Graph 2: Survival time of choleratoxinintoxicated mice treated with
anti-choleratoxin IgY antibody.

Figure 3: Intestinal mucosa of CTintoxicated mice treated with anti-CT IgY;
immuno-histochemical staining with
anti-choleratoxin antibody,
X400: choleratoxin (+).

The results presented in graph 2 showed
that survival time of mice in group 2
(CT-intoxicated mice treated with NaCl
0.9%) and in group 3 (CT-intoxicated mice
treated with IgY against Freund adjuvant,
i.e IgY isolated from egg's yolk laid of hens

immunized with Freund adjuvant only)
was similar; additionally, the survival rates
of mice in both groups at 66 hours
following CT infection were 0%. These
results indicated that a "normal" IgY (i.e
non-specifically CT-immunized IgY) had
no therapeutic effect on CT infection.
Meanwhile, in group 4 (CY-intoxicated
mice, treated with anti-CT IgY), survival time
of mice was significantly higher compared
to those in groups 2 and 3 (p < 0.001).
The results indicated that IgY specific
to CT administered gastrointestinally
possessed a therapeutic effect on CTintoxicated suckling mice. This effect was
156

Importantly, pathohistological images
of intestinal mucosa from CT-intubated
mice treated with anti-CT IgY showed little
epithelial damages compared to that of
control (figure 3).
Hirai K (2010) demonstrated that IgY
against choleratoxin B (CT-B) subunit
exerted therapeutic effect on suckling
mice infected with live V. cholerae. Together
with our findings in this study, it was
suggested that anti-CT IgY used in our
study bound to CT-B subunit on the CT
molecule, therefore inhibited the binding
of CT (or CT-B subunit) to its GM1 receptor

on intestinal mucosa's epithelial cell. Further
investigation is needed to elucidate the
mechanism of action of anti-CT IgY in
protecting mice intoxicated with CT.


Journal of military pharmaco-medicine no1-2018
CONCLUSIONS
Single injection of 0.05 mL choleratoxin
(at concentration of 1 mg/mL) through
esophageal intubation caused cholera-like
disease in suckling mice (3 - 5 days old).
In choleratoxin-intoxicated suckling mice,
a single administration of anti-choleratoxin
IgY gastrointestinally after infection can
protect choleratoxin-intoxicated suckling
mice from the toxic effect.
REFERENCES
1. Hoang Trung Kien; Do Khac Dai;
Nguyen Ngoc Tuan et al. Production of
antibody to Edwardsiella ictaluri the causative
pathogen of liver pus disease in catfish by
chicken IgY technology. Journal Information of
Pharmaco-medicine. 2010, 3, pp.71-76.
2. Tim Sunnary, Do Minh Trung, Le Thu
Hong, Lo Van Dong. Effect of IgY antibody
against Pseudomonas aeruginosa in vivo on
Pseudomonas aeruginosa-infected experimental

burn lesions. Journal of Military PharmacoMedicine. 2011, 8, pp.44-49.

3. Kazuyuki Hirai, Hideyuki Arimitsu, Koji
Umeda et al. Passive oral immunization by
egg yolk immunoglobulin (IgY) to Vibrio
cholerae effectively prevents cholera. Acta
Medica Okayama. 2010, 64 (3), pp.163-170.
4. World Health Organization. Cholera,
2015. Weekly Epidemiological Record. 2016,
38 (91), pp.433-440.
5. World Health Organization. Cholera,
2014. Weekly Epidemiological Record. 2015,
40 (90), pp.517-528.
6. World Health Organization. Cholera,
2013. Weekly Epidemiological Record. 2014,
31 (89), pp.345-356.
7. World Health Organization. Cholera,
2012. Weekly Epidemiological Record. 2013,
31 (88), pp.321-336.
8. World Health Organization. Cholera,
2011. Weekly Epidemiological Record. 2012,
No 31-32, 87, pp.289-304.

157