Experimental Parasitology 157 (2015) 23e29

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Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr

Evaluation of Echinostoma liei worm, metacercaria and redia antigens

for schistosomiasis control
G. Abdel-Monaem a, A. Farid a, *, I. Rabia b, A. El-Amir a
a
b

Zoology Dept., Faculty of Science, Cairo University, Giza, Egypt

Parasitology Dept., Theodore Bilharz Research Institute, Giza, Egypt

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

Antigens fromEchinostoma liei were

puried
using
CNBr-activated
Sepharose column.

Then used for immunization of mice
prior to infection with Schistosomiasis
mansoni.

Worm burden, eggs and oogram
count was signicantly reduced and
that was reected in normalization of
liver architecture.

E. liei worm antigens induced the
best reduction in worm burden and
tissue egg load and acted as a good
stimulator for Igs secretion.

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 20 June 2014
Received in revised form
12 May 2015
Accepted 15 June 2015
Available online 23 June 2015

While chemotherepeutic drugs, such as praziquantel, oxamniquine and metrifonate, are currently
considered safe and effective drugs for schistosomiasis treatment, reinfection occurs frequently after
drug treatment. Thus, a vaccine is sought to provide long-term treatment. Antigens from worm, metacercaria and redia of Echinostoma liei (E. liei) were puried using CNBr-activated Sepharose column, then
used for immunization of mice prior to infection with Schistosomiasis mansoni. Worm burden, hepatic
and intestinal eggs and oogram count was signicantly reduced and that was reected in normalization
of liver architecture. This referred to a signicant increase in the tested immunoglobulin level (IgM, IgG1
and IgG2).
2015 Elsevier Inc. All rights reserved.

Keywords:
Schistosomiasis
PZQ
Echinostoma liei
Worm burden
Oogram
Immunoglobulins

1. Introduction

* Corresponding author.
E-mail address: alyaafarid@hotmail.com (A. Farid).
http://dx.doi.org/10.1016/j.exppara.2015.06.008
0014-4894/ 2015 Elsevier Inc. All rights reserved.

Schistosomiasis or snail fever is one of the most signicant

parasitic diseases which have plagued humans since the time of
ancient Egyptian and Chinese mummies (Nunn and Tapp, 2000). Its
rank is high among parasitic diseases in terms of socioeconomic
and public health importance in tropical and subtropical areas

24

G. Abdel-Monaem et al. / Experimental Parasitology 157 (2015) 23e29

(Ferrari et al., 2003; Tan and Ahana, 2007).

Praziquental (PZQ), a highly active acylatedquinoline-pyrazine
against all Schistosoma species, is now universally employed as
recommended by the WHO for either individual or mass treatment
(Anonymous, 2002; Doenhoff et al., 2009). It is revolutionary
because it could be administered orally in a single dose and had
very few unwanted side effects (Doenhoff and Pica, 2006). It is
effective against cercariae, very young schistosomula and adult
worms but does not kill 3e21 day old schistosomula (the migrating
larvae) (Ross et al., 2002). Despite considerable efforts, the PZQ
binding site on the schistosome surface and the precise mechanism
of action are not yet known (Greenberg, 2005; Pica-Mattoccia et al.,
2008). Concerns about reliance on a single drug to treat 200 million
people include potential drug resistance (Keiser and Utzinger,
2007; Silva et al., 2004) and rapid reinfection rate, as people must
be retreated on an annual or semiannual basis. Thus, identication
of alternative schistosomiasis chemotherapies has become a high
priority (Abdulla et al., 2007; Doenhoff et al., 2008).
Vaccination can be targeted towards either the prevention of
infection or the reduction of parasite fecundity. Schistosome eggs
being responsible for both pathology and transmission, a vaccine
targeted toward parasite fecundity and egg viability appears,
therefore, entirely relevant (Capron et al., 2002). There is potentially numerous and promising vaccine antigens from Schistosomiasis mansoni (Sm TSP-1, Sm TSP-2, Sm 29, Sm 23, Sm-p 80, Sm 14,
Sm28-SGT, Sm28-TPI, Sm97 paramyosin and CT-COD) and, to a
lesser extent, Schistosoma haematobium, only one vaccine, namely
BILHVAX, or the 28-kDa GST from S. haematobium, has entered
clinical trials (Capron et al., 2005).
The current Schistosoma vaccine candidates may prove not to be
the most effective vaccine. It is important to identify new target
antigens and to explore alternative vaccination strategies to
improve vaccine efcacy. The available schistosome antigens and
prototype vaccine formulations induce 40e50% protection in animals, at best, using the standard readouts of reduced worm burden
or egg production and viability (McManus and Loukas, 2008).
Because larval Echinostoma liei has the upper hand when
competing with S. mansoni miracidia to attack the snail host, this
led to further study of any expected potential protective effect of
puried antigens from different stages of E. liei against murine
S. mansoni infection.

2. Materials and methods

2.1. Animals
Six to eight week old female albino CD1 mice (24 2g) bred and
maintained at the Schistosome Biological Supply Program, Theodor
Bilharz Research Institute, Giza, Egypt (SBSP/TBRI). Mice were kept
under standard laboratory care (21  C, 45e55% humidity), ltered
drinking water, 24% protein and 4% fat diet. Animal experiments
have been carried out according to the internationally valid
guidelines and ethical conditions (Nessim et al., 2000).

gp
gp
gp
gp
gp
gp
gp
gp

A:
B:
C:
CI:
D:
DI:
E:
EI:

Normal healthy control group

S. mansoni infected group
Mice were immunized with E.
Mice were immunized with E.
Mice were immunized with E.
Mice were immunized with E.
Mice were immunized with E.
Mice were immunized with E.

Iiei
Iiei
Iiei
Iiei
Iiei
Iiei

2.2. Parasites
2.2.1. S. mansoni cercariae
S. mansoni cercariae were provided by SBSP at TBRI. This strain
has been moved through outbred mice and Biomphalaria alexandrina snails, cared for and maintained at SBSP/TBRI.
2.2.2. E. Iiei worms, metacercaria and redia
E. Iiei was routinely maintained in medical malacology laboratory at TBRI. Encysted metacercariae and rediae of E. Iiei were
removed from the kidneys and pericardial cavities of experimentally infected Biomphalaria glabrata snails (El-Dafrawy, 1989; Fried
et al., 1997). Adult E. Iiei worms were obtained from small intestine of mice experimentally infected with E. Iiei.
2.3. Infection
S. mansoni infection was performed by subcutaneous injection
(s.c.) of 120 cercariae/mouse (Stirewalt and Dorsery, 1974).
2.4. Purication of E. liei antigens
Antigens prepared from metacercariae, rediae and worms of
E. Iiei were puried using CNBr-activated Sepharose column according to Axen et al. (1983). Different tissues were individually
homogenized in an iced glass tissue grinder and ultra-centrifuged
at 18.000 rpm for 1 h at 4  C. Antigens were eliminated from the
supernatants by afnity chromatography (Carter and Colley,
1978). Antibody (Sigma Chem, Co., St. Louis, Mo) was linked to
CNBr-activated sepharose 4B beads (Pharmacia ne chemicals,
Piscataway, NJ) at ratio of 7 mg protein/ml of beads (Norden and
Strand, 1984). Each supernatant was passed over a 1 ml column of
antibody coupled beads. The non-adherent host antigen-free
fraction was collected and dialyzed against phosphate buffered
saline (PBS); 0.01 M, pH 7.2. The samples were sterilized by
ltration through 0.45 mm lters (Nalgene Brand Product, Sybran
corp.; Rochchester, NY) and the protein content was estimated
using Bio-Rad kit (Bradford, 1976). All procedures were carried
out at 4  C.
2.5. Immunization with E. liei antigens
Different experimental mice groups were immunized with 3
types of E. Iiei antigens individually extracted from worms, metcercariae and rediae. Each antigen (100 mg/ml) was emulsied with
Complete Freund adjuvant (CFA) and s.c injected as 1ry immunization. Then, mice were boosted at 3rd and 4thweek (wk) post
primary injection with only 50 mg/ml of antigens emulsied in
incomplete Freund adjuvant (IFA).
2.6. Experimental design
A batch of 80 mice was divided into 8 groups (gp) (10 mice
each), as follows:

worm antigen.
worm antigen. Three days later, all mice were infected with S. mansoni cercariae.
metacercaria antigen.
metacercaria antigen. Three days later, all mice were infected with S. mansoni cercariae.
redia antigen.
redia antigen. Three days later, all mice were infected with S. mansoni cercariae.

G. Abdel-Monaem et al. / Experimental Parasitology 157 (2015) 23e29

Eight weeks after infection (PI), mice were sacriced and subjected to the following parameters:2.7. Parasitological parameters
2.7.1. Worm burden
S. mansoni adult worms were harvested by perfusion (hepatic
and intestinal) according to Duvall and De Witt (1967). Following
perfusion, number and sex of the adult worms were determined.
2.7.2. Tissue egg load (liver and intestine)
At the time of perfusion, livers and pieces of small intestine were
collected to nd out the eggs per gram (EPG) of liver or intestine.
Each piece was weighed and placed in a vol. of 5% KOH solution
(BDH Chemicals), 3e10 times greater than the vol. of tissue to be
digested (Cheever, 1968). The number of ova in each tissue was
counted in 3 slides and the average number was calculated. The
EPG of liver or intestine was calculated by multiplying the average
number of counted sample by the total amount of KOH and divided
by the weight of liver or intestine in grams.
2.7.3. Percentage egg developmental stages oogram pattern
After perfusion of the sacriced animals, the small intestine was
wholly separated and transferred to a Petri dish, the intestine was
opened lengthwise and three fragments (1 cm each) were cut and
dried on a lter paper and placed between a slide and a cover slip.
One hundred eggs were counted in each fragment in 3 fragments
per animal. Viable eggs were counted in the intestine and classied
according to their stages of development into the following stages
(Pellegrino et al., 1962).

Immature ova
Stage I
Stage II
Stage III
Stage IV
Mature ova
Dead ova

25

IgGl (Binding site, Birmingham,UK) were used at a dilution of 1/

1000, 1/3000, 1/5000 and 1/500, respectively. The reaction was
read at optical density (OD) values 492 nm using an ELISA reader
Bio-Rad Microplate Reader, Richmond, CA, USA).
3. Statistical analysis
All results were expressed as mean SE and analyzed with
Microsoft excel software. Statistical analysis of results was carried out using one-way analysis of variance (ANOVA), the mean
difference between two groups was calculated using Student's t
test. P value lower than 0.05 was considered statistically
signicant.
4. Results
4.1. Parasitological parameters
4.1.1. Total worm burden
The mean total worm burden in different mice infected gps was
evaluated under several experimental conditions. Untreated
infected control (gp B) recorded 31.60 0.33. The perfusion data
(liver and porto-mesenteric) showed a very highly signicant
reduction (P < 0.001) in 3 different cases of immunizations (CI, D1
and EI gp) to be 7.10 0.29, 11.30 0.30 and 18.60 0.21, respectively, compared to infected control gp B (31.60 0.33). It was clear
that immunization with worm antigens recorded the greatest
percent reduction (PR) (77.5%) followed by immunization with
metacercaria and redia antigens (64.2% and 41%, respectively)
(Table 1).

Embryo occupies one-third the diameter of the egg.

Embryo occupies one-half the diameter of the egg.
Embryo occupies two-thirds the diameter of the egg.
Embryo occupies the entire egg shell.
It contains a fully developed miracidium inside the egg shell.
It appears semitransparent granular, or darkened with retracted embryo.

2.8. Immunological parameters

4.2. Number of ova/gm tissue

2.8.1. Determination of serum-specic immunoglobulins

Total and subclasses of anti-soluble egg antigens (SEA) polyvalent mouse immunoglobulins (Igs) were measured using indirect
ELISA based on the method of Engvall and Perlman (1971). To
measure polyvalent Igs, IgG, IgM and IgGl; ELISA microtitre plates
were coated with 250mg/well of 30 mg/ml of SEA. Sera were diluted
1:100 for measurement of polyvalent Igs and IgG, and diluted 1:100
for measurement of IgM or IgGl. Peroxidase conjugate, rabbit antimouse Igs (Sigma) and monoclonal sheep anti-mouse IgG, IgM and

4.2.1. Number of ova/gm hepatic tissue

The number of ova/gm hepatic tissue of the infected untreated
control mice (gp B) was 3011 374. These data show that the immunization of mice with 3 different E. liei antigens (worms, metacercariae and redia antigens) recorded a high signicant decline in
total count of eggs with (P < 0.001) as gp CI, DI and EI (988 277,
1158 33 and 812 90, respectively). As depicted in Table 2, the
immunization of the infected mice with rediae antigens (gp EI)
resulted in a highest signicant reduction in reference to the other

Table 1
Effect of immunization with E. liei (worm, redia and metacercaria antigens) on worm burden picture in S. mansoni-infected mice.
Worm burden (mean SE)

Mice group

Male
gp
gp
gp
gp

B
C1
D1
E1

S.
S.
S.
S.

mansoni
mansoni
mansoni
mansoni

infected
infected
infected
infected

Control
mice E. liei worm Ag
mice E. liei metacercariae Ag
mice E. liei redia Ag

9.40
1.00
2.10
2.40

Total worm burden

Female

0.30
0.35
0.23
0.11

*** Statistically signicant difference at P < 0.001 compared to infected control gp.
SE: Standard error.

12.20
2.10
3.20
6.20

PR (%)

Couples
0.40
0.38
0.43
0.42

5.10
2.00
3.00
5.00

0.29
0.31
0.25
0.17

31.60
7.10
11.30
18.60

0.33
0.29***
0.30***
0.21***

e
77.5%
64.2%
41.1%

26

G. Abdel-Monaem et al. / Experimental Parasitology 157 (2015) 23e29

Table 2
Effect of immunization with E. liei (worm, redia and metacercaria antigens) on tissue egg load in S. mansoni infected mice.
Ova count (mean SE)

Mice group

gp
gp
gp
gp

B
C1
D1
E1

S.
S.
S.
S.

mansoni
mansoni
mansoni
mansoni

infected
infected
infected
infected

Control
mice E. liei worm Ag
mice E. liei metacercariae Ag
mice E. liei redia Ag

Liver

PR

Intestine

PR

3011 374
988 277***
1158 33***
812 90***

e
67.3%
61.5%
73.1%

17234 1291
4158 234***
6121 133***
3889 303***

e
75.9%
64.2%
77.4%

*** Statistically signicant difference at P < 0.001 compared to infected control gp.
SE: Standard error.

Table 3
Effect of immunization with E. liei (worm, redia and metacercaria antigens) on oogram pattern of S. mansoni infected mice.
Egg developmental stages (M SE)

Mice group

Immature ova
gp
gp
gp
gp

B
C1
D1
E1

S.
S.
S.
S.

mansoni
mansoni
mansoni
mansoni

infected
infected
infected
infected

Control
mice E. liei worm Ag
mice E. liei metacercariae Ag
mice E. liei redia Ag

67.2
47.9
4.9
22.1

5.0
3.9
5.4***
3.8**

Mature ova
33.1
29.9
3.3
40.8

2.6
3.1
1.4***
3.0

Dead ova
1.7
22.2
91.8
37.1

0.3
1.1*
7.4***
2.0**

* Statistically signicant difference at P < 0.05 compared to infected control gp.

** Statistically signicant difference at P < 0.01 compared to infected control gp.
*** Statistically signicant difference at P < 0.001 compared to infected control gp.
SE: Standard error.

gps (CI and DI) especially to the infected control (gp B) with PR:
73.1%, 67.3% and 61.5%, respectively (Table 2).
4.2.2. Number of ova/gm intestinal tissue
The mean total number of intestinal egg load in the infected
control mice (gp B) recorded 17234 1291. Immunization of mice
with antigens of different stages of E. liei (worms, metacercariae
and rediae) induced a high signicant PR (P < 0.001) in the
number of intestinal egg load which recorded 4158 234,
6121 133 and 3889 303, respectively (Table 2). Comparing the
ova count number next to different immunizations, it induced a
very high and signicant PR (P < 0.001) when the infected mice
were immunized with redia and worms antigens (gp EI and CI)
(77.4 and 75.9%, respectively) in reference to the infected control
(gp B). But, immunization of infected mice with metacercariae
antigens (gp DI) induced only 64.2% PR of the ova/gm intestinal
tissue (Table 2).
4.3. Percentage of various egg developmental stages
In the infected untreated mice (gp B), the number of the dead
ova is very small (1.7 0.3) and much less than that of immature
ova (67.2 5.0) and mature ova (33.1 2.6). It is clear from Table 3
that immunization with E. liei worms antigens (gp CI) caused a
signicant increase in ova death to be 22.2 1.1, whereas there
was no effective change in immature and mature eggs no.
(47.9 3.9 and 29.9 3.1, respectively). Next, redia antigens (gp
EI) stimulated a noticeable signicant decreased (P < 0.01) in
immature ova no. (22.1 3.80) and the no. of dead ova was
signicantly increased (P < 0.01) (37.1 2.00), while it had no
effect on the mature ova no. (40.8 3.0). Surprisingly, E. liei
metacercariae antigens (gp DI) had a promising effect on the dead
ova no. (91.8 7.4) as highly and signicantly (P < 0.001)
increasing and highly and signicantly (P < 0.001) decreasing the
no. of both immature and mature ova count (4.9 5.4 and
3.3 1.4, respectively).
All these data were statistically calculated in comparison to ova

no. of infected control (gp B).

5. Immunological parameters (serum immunoglobulin
measurement)
5.1. IgM level
The normal IgM level measured in sera of healthy unimmunized mice (gp A) recorded 0.24 0.20 while in infected control
mice (gp B) it became 0.43 0.16. Only immunization of normal
mice with different E. Liei antigens (gp C, D and E) induced an
increase in IgM level (0.39 0.16, 0.33 0.19, and 0.32 0.22,
respectively). On the other hand, as well known previously that
infection induced antibody secretion. So, infected mice immunized with E. liei different antigens recorded a signicant
(P < 0.001) increase according to the type of immunizing antigens
and began with worms (gp CI), metacercariae (gp DI) and rediae
(gp EI) antigens as the following 0.50 0.18, 0.52 0.20 and
0.62 0.13, respectively, indicating the highest stimulation of
rediae antigens as immunostimulant in case of S. mansoni infection (Table 4).
5.2. IgG1 level
In case of gp A, the IgG1 level recorded 0.20 0.18, but when
mice were infected with S. mansoni cercariae (gp B) the IgG1,
level was highly increased (0.54 0.10). The recorded data
shown in (Table 4) illustrate that only immunization with
different E. liei stages antigens [worms (gp C), metacercariae (gp
D) and rediae (gp E)] induced an increase in IgG1 level to be
0.36 0.19, 0.59 0.14 and 0.36 0.20, respectively, while, the
same antigens when injected into S. mansoni infected mice,
induced more signicance (P < 0.05) elevation in IgG1 level as
0.87 0.23 (gr CI), 0.78 0.19 (gr DI) and 0.81 0.21 (gr EI),
respectively.
It was obvious that the increase in IgG1 level according to immunization alone or with infection referring to normal or infected

G. Abdel-Monaem et al. / Experimental Parasitology 157 (2015) 23e29

27

Table 4
Effect of immunization with different stages of E. liei (worm, redia, metacercaria) antigens on the serum immunoglobulin level of S. mansoni infected mice.
IgM (X SE)

Mice group
gp
gp
gp
gp
gp
gp
gp
gp

A
B
C
C1
D
D1
E
E1

Normal control
Infected Control
Normal mice Worm Ag
Infected mice Worm Ag
Normal mice Metacercariae Ag
Infected mice Metacercariae Ag
Normal mice Redia Ag
Infected mice Redia Ag

0.24
0.43
0.39
0.50
0.33
0.52
0.32
0.62

0.20
0.16
0.16
0.18***
0.19
0.20***
0.22
0.13***

IgG1 (X SE)
0.20
0.54
0.36
0.87
0.59
0.78
0.36
0.81

0.18
0.10
0.19
0.23**
0.14
0.19**
0.20
0.21**

IgG2 (X SE)
0.29
0.71
0.40
1.18
0.70
1.00
0.41
1.08

0.21
0.21
0.14
0.15*
0.17
0.23**
0.21
0.14**

* Statistically signicant difference at P < 0.01 compared to infected control gp.

** Statistically signicant difference at P < 0.05 compared to infected control gp.
*** Statistically signicant difference at P < 0.001 compared to infected control gp.
SE: Standard error.
X: Mean OD reading at 492 nm.

gps are nearly the same for each direction and there is no effect on
the type of immunizing antigens.
5.3. IgG2 level
The recoded level of IgG2 in normal mice (gp A) was 0.29 0.21,
this data was highly increased when mice were infected with
S. mansoni (gp B) to be 0.71 0.21. Similar to the data of IgG1, the
immunization with different types of E. liei antigens prepared from
different life cycle stages [worms (gp C), metacercariae (gp D) and
rediae (gp E)], there is an increase in IgG2 level (0.40 0.14,
0.70 0.17 and 0.41 0.21, respectively), whereas infection with
S. mansoni in addition to immunization stimulated a vigorous
(P < 0.01) increase in IgG2 level to record 1.18 0.15 (gp CI),
1.00 0.23 (gp DI) and 1.08 0.14 (gp EI), respectively (Table 4). The
increase in IgG2 level is not specically due to only one antigen, but
nearly the same is true for all 3 types of antigens.
6. Discussion
Schistosomiasis is the second most signicant parasitic disease
in the world after malaria in terms of sosioeconomic and public
health importance. It is estimated that 207 million people are
infected in 74 countries and more than 779 million people are at
risk of infection, with mortality estimated up to 280 thousand
deaths annually in sub-Saharan Africa alone (Utzinger et al., 2003).
The WHO strategy for schistosomiasis control focuses on
reducing disease through periodic, targeted treatment with PZQ.
This involves regular treatment of all people in at-risk groups.
Treatment should be complemented with health education, as well
as access to safe water and good sanitation (WHO, 2010).
After two decades of widespread chemotherapy with safe and
effective drugs, drug treatment and other existing control measures
have failed to eliminate the incidence of infection, morbidity and
mortality due to schistosomiasis infection. Additionally, schistosomiasis still spread into new areas (McManus, 2005). Generally,
severe symptoms of the disease appear only in people who harbor
large numbers of parasites, so if a vaccine is effective in reducing a
patient's burden of worms by 50% or more, this will be reected in a
reduction in the number of eggs retained in the tissues, and, thus, it
can dramatically reduce the number of severe cases of the disease
and morbidity (Siddiqui et al., 2007, 2011). The development of an
effective vaccine against schistosome is a critical stage despite the
fact that progress has been relatively slow, and the successful use of
animals with attenuated vaccines combined with recent recombinant derived Schistosoma antigens. Partial immunity developed
against schistosomiasis in individuals living in endemic areas
suggests that the development of a safe and effective vaccine is

possible (McManus and Loukas, 2008).

Echinostoma species develop their larval stages in B. alexandrina
snails, the most important intermediate host of S. mansoni (Loker
and Adema, 1995). The host-parasite relationship between E. liei
and its natural intermediate host B. alexandrina would provide
considerable help for effectively studying this potential biocontrol
agent and elaborate methods to utilize it in the control of and
protection against schistosomiasis (El-Dafrawy et al., 2001;
Sandland et al., 2007).
The aim of this work was designed to investigate the possible
effect of immunization protocol against S. mansoni infection with
puried antigens from E. liei on resistance to infection and associated immunoparasitological changes in murine experimental
model. Some experimental studies described the effects of interactions between Echinostoma caproni and S. mansoni in homologous or heterologous infection of denitive hosts. Mice preinfected by E. caproni showed increase in natural resistance, with
decrease of the S. mansoni load in experimental conditions
(Christensen et al., 1987, 1988). Echinostomes have been proposed
as potential bio-control agents for medically important schistosomiasis disease, depending on the fact that E. liei has the ability of
blocking subsequent infection of the snail B. alexandrina by Schislosoma miracidiae (El-Dafrawy et al., 2001; Loker and Adema,
1995).
In this study, it was clear that immunization with worm antigens has the greatest effect on the total worm burden of infected
mice as PR 77.5% followed by metacercariae then rediae antigens
(64.2% and 41.1%, respectively).
These results were in agreement with Rabia et al. (2007), who
studied the effect of immunization protocol against S. mansoni
infection with puried metacercarial antigens from E. liei alone or
combined with BCG on resistance to S. mansoni infection and
associated immunoparasitological changes in murine experimental
model. The results have revealed a highly signicant reduction in
worm burden of the two immunized gps (73.5% and 78.2%,
respectively) compared to the infected control gp (P < 0.001).
Also, Kobia et al. (2012) tested the effect of immunizing Swiss
white mice with soluble proteins prepared from the digestive gland
(DG) and the rest of the snail body (RT) of intermediate host,
B. pfeiferri. The immunized mice were challenged with S. mansoni,
worm reduction, and gross pathology analyzed. Worm reduction in
RT was 60.5% while that of DG was 43.3%. On the other hand,
Martins et al. (2012) found that immunization of mice with GPIanchored proteins induced a 42% reduction in worm burden, 45%
reduction in eggs per gram of hepatic tissue, 29% reduction in the
number of granulomas per area, and 53% reduction in the granuloma brosis. Taken together, these data support the potential of
surface-exposed GPI-anchored antigens from the S. mansoni

28

G. Abdel-Monaem et al. / Experimental Parasitology 157 (2015) 23e29

tegument as vaccine candidate.

Previous studies showed that radiation-attenuated vaccine
gives protection levels for S. mansoni in host various species. Reda
et al. (2012) evaluated the effect of various vaccination strategies
in C57BL/6 mice, including single or multiple vaccination strategy,
subcurative dose (20 mg/kg) of PZQ, and a combination of single
vaccination with subcurative dose of PZQ. Treatment either with
subcurative dose of PZQ or with a single vaccination of attenuated
cercariae (500 per mouse) caused a signicant reduction in total
worm burden, hepatic and intestinal ova counts 43.0%, 73.2%, 59.5%
and 37.9%, 52.0%, 26.3%, respectively. However, the multiple vaccination strategy resulted in much higher reduction in total worm
burden, hepatic and intestinal ova count. They also reported that
multiple vaccination strategy resulted in high reduction of worm
burden, hepatic and intestinal ova counts 72.5%, 90.7%, 65.7%,
respectively.
The number of ova/gm tissue is also an important criterion for
the evaluation of the effect of vaccine on the infection (Mostafa,
2005). Rabia et al. (2007) observed a signicant reduction in hepatic and intestinal tissue egg loads in response to vaccination with
puried metacercarial antigens from E. liei alone or combined with
BCG.
This study has shown that immunization of mice with E. liei
worms and rediae antigens achieved the best PR in ova count in
both liver and intestine. However, redia antigens recorded high PR
in no. of ova/gm hepatic and intestinal tissues (73.1% and 77.4%,
respectively). This was followed by worm antigens (67.3% for hepatic tissue and 75.9% for intestinal tissue).
Hamed et al. (2010) evaluated the nucleoprotein extracted from
either susceptible or resistant B. alexandrina snails to protect
against schistosomiasis. The vaccination schedule consisted of a
subcutaneous injection of 50 mg protein of each antigen followed by
another inoculation 15 days later. The nucleoprotein of susceptible
snails showed reduction in worm and ova counts by 70.96% and
51.31%, respectively, whereas the nucleoprotein of resistant snails
showed reductions of 9.67% and 16.77%, respectively.
At the beginning, the changes in the oogram of intestinal fragments were considered signicant when one or more developmental stages of immature as well as mature eggs disappear
(Pellegrino et al., 1967). In the present work, with regard to the
infected untreated mice (gp B), the number of dead ova (1.7 0.3)
was much less than that of immature ova (67.2 5.0) and mature
ova (33.1 2.6). The best results were noticed in immunization of
infected mice with metacercaria antigen, where the no. of dead ova
(91.8 7.4) increased and the no. of both immature and mature ova
(4.9 5.4 and 3.3 1.4, respectively) were decreased. These results
were coincided with the results of Rabia et al. (2007); they reported
an increase in the number of dead ova after immunization with
E. liei metacercarial antigens alone or combined with BCG. Recently,
Saber et al. (2013) conrmed that a good vaccine should achieve
some parameters as reduction in worm burden, tissue egg load and
a changing in oogram pattern. They evaluated the efcacy of
fructose-1, 6-bis phosphate aldolase (SMALDO) DNA vaccination
against S. mansoni infection using different routes of injection. Both
i.p. and s.c. vaccination routes resulted in a signicant reduction in
worm burden (46.2% and 28.9%, respectively, P < 0.01). This was
accompanied by a signicant reduction in hepatic and intestinal
egg counts (41.7% and 40.2%, respectively, P < 0.01) in the i.p.
injected gp only. The number of dead eggs was signicantly
increased in both i.p and i.m gps (P < 0.01). High anti-SMALDO IgG
antibody titers were detected in sera of all vaccinated gps (P < 0.01)
compared to the control gp.
Antibodies play a key role in different effector or regulatory
functions depending on the isotypes activation. After egg lying,
high titers of antibodies specic to carbohydrate epitopes are

detected in plasma of both mice and human (Omer-Ali et al., 1988).

In mice, the passive transfer of these antibodies greatly increases
the size of granuloma (Simpson et al., 1990). Numerous studies
showed that glycosylated schistosomal antigens constitute the
major target for host antibody response (Nyame et al., 2003). IgM
and IgG antibodies develop when schistosome worms become
mature. Studies on baboons infected with S. mansoni found that
IgM antibodies appear 3 wk PI, peaked 7 wk later and then decline
rapidly to low but persistent levels. IgG antibodies appear 5e6,
peaked 10e13 wk PI and persist undiminished (Suzuki and Damian,
1981). Botros et al. (1995) and Hassanein et al. (1997) reported that
immunization procedure probably generated memory B cells, then
on exposure to infection boosted secondary immune responses,
which was higher (IgGl, IgG and IgM) than infected control. Wynn
et al. (1996) reported that vaccinated mice showed signicant increases in parasite-specic antibodies in particular IgGl isotype, and
developed macrophages with increased nitric oxide-dependent
killing activity against the parasites.
Our results revealed that immunization with E. liei worm antigens stimulated IgG1 and IgG2 secretion more than any other type
of antigens. On the other hand, E. liei rediae antigens were more
effective than other types in stimulating IgM secretion.
Eberl et al. (2001) reported that the radiation-attenuated
S. mansoni vaccine was highly effective in rodents and primates.
Also, vaccination induced parasite-specic IgM and IgG, which
reached high levels at the time of challenge, while in control animals levels did not raise markedly before egg deposition. Also,
Rabia et al. (2007) conrmed the signicant increase of specic
antibodies IgG and IgM together with higher signicant increase in
IgGl which was associated with great reduction in ova count and
worm burden.
Successful vaccine has four effects: Reduction in adult worm
numbers, egg production, protection against acute schistosomiasis
and therapeutic effect on adult worms (Ahmad et al., 2011).
In conclusion, E. liei worm antigens induced the best reduction
in worm burden and tissue egg load and acted as a good stimulator
for Igs secretion in S. mansoni infected mice. Such ndings suggest
that E. liei worm antigens can be used in protecting mice against
schistosmasis.
Acknowledge
The authors gratefully acknowledge the use of the facilities of
SBSP in TBRI, Giza, Egypt. I much appreciate the great effort of the
staff of medical malacology laboratory for providing different life
cycle stages of E. liei.
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