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Toxocara vitulorum is a nematode parasite of small intestine of buffalo, particularly young age calves, causing high morbidity and mortality. The control of this infection produces considerable eco-nomic benefits. Immunization of Wistar rats with different T. vitulorum antigens; (execratory/ secreto-ry antigen (ESAg), peri-enteric fluid antigen (peAg) and embryonated eggs antigen (EEAg)) were evaluated for induction of protection against a challenge oral infection with embryonated eggs of T. vitulorum. It was found that peAg induce the highest protection level (100%) followed by ESAg (96.4%-97.5%) then EEAg (93.7%-96.7%). Mean number of larvae extracted from immunized rats was significantly (P≤ 0.05) lower than that extracted from the control non-immunized one. The num-ber of larvae collected after digestion of the liver at 3rd day post challenge (dp.c.) was decreased from 998.5 in control rats to 26 and 41.5 in rats immunized with ESAg and EEAg respectively. In the same time no significant (P≤0.05) difference in size of larvae extracted from immunized and control posi-tive rats. For conclusion, the study demonstrated the value of rats as experimental model for investiga-tion T. vitulorum infection. It characterized PeAg as a valuable immunogenic and protective antigen in minimize the infection by T. vitulorum between mother and calves in infected farms.
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293
Journal of the Egyptian Society of Parasitology, Vol. 47, No. 2, August
2017 J. Egypt. Soc. Parasitol. (JESP), 47(2), 2017: 293 - 301
EVALUATION OF THE PROTECTIVE VALUE OF SOME TOXOCARA
VITULORUM ANTIGENS IN RATS By
OLFAT A. MAHDY*, WAHEED M. MOUSA, SAHAR Z. ABDEL-MAOGOOD
AND SHIMAA ABDEL-RADI
Department of Parasitology, Faculty of Veterinary Medicine, Cairo University,
Giza, Egypt (*Correspondence: E-mail: dr.olfat.mahdy@cu.edu.eg)
Abstract
Toxocara vitulorum is a nematode parasite of small intestine of buffalo, particularly young age
calves, causing high morbidity and mortality. The control of this infection produces considerable eco-
nomic benefits. Immunization of Wistar rats with different T. vitulorum antigens; (execratory/ secreto-
ry antigen (ESAg), peri-enteric fluid antigen (peAg) and embryonated eggs antigen (EEAg)) were
evaluated for induction of protection against a challenge oral infection with embryonated eggs of T.
vitulorum. It was found that peAg induce the highest protection level (100%) followed by ESAg
(96.4%-97.5%) then EEAg (93.7%-96.7%). Mean number of larvae extracted from immunized rats
was significantly (P≤ 0.05) lower than that extracted from the control non-immunized one. The num-
ber of larvae collected after digestion of the liver at 3rd day post challenge (dp.c.) was decreased from
998.5 in control rats to 26 and 41.5 in rats immunized with ESAg and EEAg respectively. In the same
time no significant (P≤0.05) difference in size of larvae extracted from immunized and control posi-
tive rats. For conclusion, the study demonstrated the value of rats as experimental model for investiga-
tion T. vitulorum infection. It characterized PeAg as a valuable immunogenic and protective antigen in
minimize the infection by T. vitulorum between mother and calves in infected farms.
Key words: Toxocara vitulorum, Rats, Experimental study, ELISA
Introduction
Toxocara vitulorum is a nematode parasite
of small intestine of buffaloes, particularly
young age calves, causing high morbidity
and mortality. The larvae remain in the tis-
sues of the infected dam until just prior to
parturition then migrate to the mammary
gland and milk for subsequent ingestion by
the calf (Starke-Buzetti and Ferreira, 2006).
Migration of the larvae through tissues pro-
duced severe inflammatory reactions and
consequently to a wide range of pathological
and clinical manifestations (Strube et al,
2013). The newly born calves were found to
be highly infected at age of 15-90 days and
consequently act as a source of infection to
other animals in the farm as infected calves
started to shed eggs at 16-23 days of age
(Raza et al, 2013). The economic level and
poor knowledge of farmers in tropical and
sub- tropical countries which exposed to
drink raw milk are likely to accelerate the
transmission of the parasite causing larval
migraines in human body (Rast et al, 2013).
Immunization trials with different T. vi-
tulorum antigens using different experi-
mental animals was investigated by some
authors. Amerasinghe et al. (1992) reported
that PeAg induced in mice 100% protection
in comparison with ESAg which produce
slight lower protection (82.6%). Also vac-
cination of rabbits by EEAg decreased tis-
sues invasion ability of larvae (El- Askalany
et al, 2008).
With the presence of VLM infection in the
Islamic Countries (low contact to dogs), T.
vitullorum was considered as probable cause
for the disease in human. Several stud-ies
are available on other Toxocara spp. causing
VLM but some still under investiga-tion for
T. vitulorum such as its migratory pattern in
both definitive and other hosts, duration of
its larvae as inhibited larva in final hosts and
the role of immunization in prevention of
infection. Moreover, the buf-faloes
constitute one of the most important features
of the Livestock as they occupy the
prominent position among farm animals and
play a great part in the national economy in
Egypt. Several experimental animals, inclu-
ding Guinea-pigs (Buijs et al, 1993), rabbits
(El-Askalany et al, 2008), mice (Cardillo et
294
al, 2009), rats (El Kabany, 2013) and pigeo-
ns (Rahbar et al, 2013) in studying toxocari-
asis. But, the role of rats as experimental
model for investigation of T. vitulorum in-
fection was neglected in comparison to other
laboratory animals (Strub et al, 2013).
This study aimed to investigate the effica-
cy of vaccination by different T. vitulorum
antigens in animal protection from infection
using rats as a model for the present study to
demonstrate its value in investigation of T.
vitulorum infection.
Materials and Methods
All the study steps and procedures were
approved by the Institutional Animal Care
and Use Ethical Committee (CU-IACUC) of
Cairo University (CU/ II/ S/ 2016/ 2017).
Antigens of T. vitulorum: Intact, active T.
vitulorum adult worms were collected from
intestine of freshly slaughtered buffalo
calves in Cairo abattoir, Egypt. The worms
were washed in normal saline then used for
preparation of ESAg and peAg while T.
vitulorum eggs were collected from the
distal part of the uteri of the adult female
worms and then embryonated (Starke and
Ferreira, 2006) to be used to prepare EEAg
and induction of infection of rats.
Adult T. vitulorum ESAg: The ESAg was
collected after incubation of both adult ac-
tive male and female T. vitulorum at 37oC
for 2 hrs in PBS supplemented with antibi-
otic (Amerasinghe et al, 1992). Then the
fluid was centrifuged at 460xg for 5 minutes
and the supernatant was filtered. Protein
content was increased by concentration us-
ing polyethylene glycol in molecular porous
membrane tubing 6-8 MW cut off (Spectrum
Medical Inc., Los Angeles, CA 900060) at
4°C (Goswami et al, 2013). The concentrat-
ed fluid was collected and its protein content
was measured using Lowry's Assay (1951)
then stored at -20°C till use.
Adult T. vitulorum PeAg: The PeAg was
collected from adult active T. vitulorum by
puncturing the posterior end of each parasite
with needle and perienteric fluid was
drained, collected and centrifuged at 460xg
for 5 minutes then treated as ESAg (Ferreira
and Starke-Buzetti, 2005).
Toxocara vitulorum EEAg: The EEAg was
prepared from the previous embryonated T.
vitulorum eggs after several washing with
0.01 PBS (PH 7.4) by centrifugation at 1500
r.p.m. for 3 minutes. The sedimented eggs
were mixed with an equal volume of the
same solution then homogenized at 6000
r.p.m for 5 minutes in an ice bath. The ho-
mogenized sample was sonicated for 5
minutes at 5 pulse rate and 60-80 amplitude
value using Coleparmer Ultrasonic homoge-
nizer then suspension was subjected to high
speed centrifugation at 14,000 rpm for 30
minutes at 4ºC and the supernatant was sep-
arated as EEAg, concentrated and its protein
contents was measured and stored as before
(El- Askalany et al, 2008).
Experimental study: Sixty (60) Male
Wistar white rats of 120-200 gram were di-
vided into 4 groups; GA: (immunized T. vi-
tulorum group) 30 rats were immunized with
different T. vitulorum antigen (ten rats for
each type of antigen), GB: (control posi-tive
T. vitulorum group) ten non immunized rats
infected with embryonated T. vitulorum eggs
at time of challenge infection, GC: (ad-
juvant control group) ten rats were injected
with adjuvant alone and GD: (control nega-
tive group) ten non immunized rats kept non
infected throughout the experimental study.
Immunization protocol: According to the
method given by Tang et al. (2015) with lit-
tle modification, the prepared T. vitulorum
antigens (ESAg, PeAg & EEAg) were used
to produce hyperimmune sera. Sera were
collected before immunization as negative
sera. Rats were immunized subcutaneously
with1.2 mg protein of T. vitulorum antigens,
emulsified 1:1 with mineral oil. After 2
weeks another 1.2 mg protein was mixed 1:1
in oil and divided into 3 doses (day 14, 21 &
28) then injected subcutaneously. One week
after the last immunization; Rats were bled
for serum collection and the level of anti-
bodies was measured. The collected sera
were stored at -20°C until used as positive
295
sera.
Infection/challenge protocol: At the end of
the immunization program and arrival of
antibodies level versus each antigen into
high level, each immunized rat was inocu-
lated by 10,000 embryonated T. vitulorum
eggs orally using stomach tube. The blood
samples were obtained by slaughtering of 2
rats at 3, 7, 15, 30 and 45 days post inocula-
tion. Rats in GD didn't receive any eggs and
slaughtered at the same previous time for
comparison and comparing the level of anti-
bodies between the immunized and control
groups using ELISA.
Table 1: Experimental design (T. vitulorum) in ten rats each
A
B
C
D
Group
Immunized
Control +ve
Adjuvant control
Control -ve
Type of antigen
ESAg
peAg
-
-
-
I
m
m
u
n
i
z
a
t
i
o
n
p
e
r
i
o
d
35 dp.im
S/C injection with 1.2 mg protein
S/C injection with
Not injected
All rats bled and level of
antibodies in sera determined by ELISA
Zero day
of each antigen with oil adjuvant
Not injected
adjuvant alone
14, 21 and 28
S/C injection
S/C injection with
Not injected
dp.im.
With 400 μg with oil adjuvant
Not injected
adjuvant alone
Post immunization program
Oral inoculation with 10000 T. vitulorum embryonated eggs
Not infected
3 dp.i./p.c.
7 dp.i./p.c.
1) Blood samples obtained by slaughtering of 2 rats and fluctuation in level of antibod-
15 dp.i./p.c.
ies between collected sera of different groups detected by ELISA.
30 dp.i./p.c.
2) Postmortem examination was performed.
45 dp.i./p.c.
*dp.i. =day post infection,*dp.c. =day post challenge, *dp.im. =day post immunization.
Post mortem inspection: Internal organs of
the sacrificed rats including livers, lungs,
kidneys, spleens, brains and muscles were
minced separately and digested in 20 times
their volume of (0.7% pepsin and 0.7% HCl
in saline) for 6 hrs at 39°C then the larvae
were sediment and counted carefully post
each slaughtering time (Barriga and Omar,
1992). The percentage of protection was cal-
culated (Amerasinghe et al, 1992) by sub-
tracting the number of obtained larvae from
immunized group from the number of larvae
found in control one divided on number of
larvae found in control rat X 100. The mor-
phology of the larvae obtained from immun-
ized and control positive rats at 7th dp.i/p.c.
were carefully inspected. The larvae were
measured using micrometer slide and micro-
meter eye Piece. Significance difference in
their number and size were evaluated.
SEM examination: Larvae were fixed in a
2.5% glutraldhyde solution in a 0.1 M sodi-
um cacodylate buffer for 4 hrs. at 4 ºC. After
two washes in the same buffer (0.2 M), the
samples were dehydrated in a graded ethanol
series, dried by critical point drying with
EMSCOPE CPD 750 and coated with gold-
palladium for 5 min at 100 min-1. The sam-
ples were then observed with a S450 scan-
ning electron microscope (Hitachi) at an ac-
celerating voltage of 15 kV.
ELISA: Fluctuation in antibodies titter in
each group was evaluated at different days
of scarification in control and immunized rat
using ELISA. The assay was performed (Liu
et al, 2015) with little modification. Optimal
dilutions of various reagents were deter-
mined using Checkerboard titration. Micro-
titration polystyrene plates, 96-well, was
coated with 4g protein/ml from each anti-
gen in coating buffer (200μl/well) then in-
cubated overnight at 4ºC. Plates were wash-
ed three times with washing buffer (0.05%
Tween-20 in PBS pH 7.3) then blocked us-
ing 0.5% bovine serum albumin in PBS-
Tween-20 (200μl/well) & incubated at 37ºC
for an hour. After washing, reagents were
added (100l/well) and incubated at 37ºC
for an hour with shaking and washing after
each step: tested and control serum samples
(1:100), Protein A horse reddish peroxidase
conjugate, (Sigma) (1:1000 in PBS). Reac-
tion was detected by adding 100μl/well of
substrate o-phenylenediamine dihydrochlo-
ride (OPD), plus H2O2for 30 minutes and
stopped by adding 50μl/well of 1N H2SO4.
296
Optical densities (O.D) were read at 450nm
with a micro-ELISA reader system. The sera
were considered positive when absorbance
values were as or more than the cut off value
that double fold of the mean negative con-
trol sera.
Statistical analysis: Data were computer-
ized and analyzed by the statistical package
SSPS by using Chi-square test. The differ-
ences were expressed as significant at P
0.05 (Verzani, 2004).
Results
Immunization of rats by different T. vitulo-
rum antigens showed marked reduction in the
total mean number of larvae extracted from
tissue of immunized rats as compared to
control non-immunized groups with dif-
ferences related to antigen type used (Tab. 2).
The mean number of extracted larvae
decreased from 998.5 at 3rd p.i. to 87 at 45th
dp.i. during migration from liver to muscles in
control positive group, from 983 to 79 in
adjuvant group, from 26 to 3 in group im-
munized by ESAg and from 41.5 to 7 in the
group immunized by EEAg in same period
between liver and muscles respectively.
After oral inoculation of (GB & GC) with
T. vitulorum embryonated eggs (Tab. 3), the
highest number of larvae was in rats’ liver
sacrificed at3 dp.i., extracted from different
organs except muscles at 7th dp.i. with high
number in lung. While at 15th dp.i., larvae
were extracted from different organs include
ing muscles till the study end with progress
sive decreasing number of larvae. The same
pattern was detected in the immunized rats
after challenge with T. vitulorum embryo-
nated eggs at 3 dp.c., but larvae were ex-
tracted only from liver, lung and spleen at
7th dp.c. in ESAg immunized rats. While in
EEAg immunized rats, larvae also extracted
from the kidney.
At 15th dp.c., larvae were extracted only
from liver of ESAg immunized rats till the
study end. Few numbers of larvae were ex-
tracted from liver, lung, spleen and muscles
of EEAg immunized rats at 30th dp.c. & 45th
dp.c., but only detected in liver.
In the present study, immunized rats by
different antigens as compared to control
positive rats after infection challenge (Tab.
4) showed 100% protection in rats immun-
ized with PeAg followed by ESAg (96.4-
97.5%), lowest was in the EEAg immunized
rats (93.7-96.7%). The results showed no
significant difference in size of extracted lar-
vae from control positive or immunized rats.
Mean size of larvae from control positive
group was (391.6±8x18.4±2.1), (399.8±16x
18.8±2.3), (413.5±11x19.6±2.6), (418±18x
18.2±3.1) & (419.3±10x19.4±2.9) from liv-
er, lung, spleen, kidney and brain respective-
ly, as corresponded with (360-440x15-18),
(360-459x16-20) and (366-468x16-20) from
liver, lung and spleen of rats immunized by
ESAg respectively and (362-442x16-18),
(362-461x16-20), (369-462x17-20) and
(368-460x17-20) from liver, lungs, spleen
and kidney of EEAg immunized rats respec-
tively. Scan Electron Microscope showed no
differences in morphology between the ex-
tracted larvae from the rat’ groups. T. vitulo-
rum larvae appeared small cylindrical with a
rather narrow anterior extremity. It was
ensheathed in a loose cuticle that forms sev-
eral wrinkles, particularly at the cervical re-
gion. The mouth opening is roughly circular
to triangular. It lies on the top of the ce-
phalic plate, and is surrounded with six mas-
sive and ill-defined lips. In some larvae, the
cuticular incisions separating these lips are
not deep (Fig. 2).
Table 2: Total mean No. of T. vitulorum larvae extracted from sacrificed rats at different days post infection & post challenge
Time of slaugh-
Total mean number of T. vitulorum larvae extracted from
tering
GA ESAg
GA EEAg
GB
GC
3 dp.i./p.c.
26
41.5
998.5
983
7 dp.i./ p.c.
22
29.5
887
880
15 dp.i/ p.c.
11
18.5
300.5
280
30 dp.i/ p.c.
5.5
9.5
150.5
123.5
45 dp.i./ p.c.
3
7
87
79
297
Table 3: Mean No. of T. vitulorum larvae extracted from 2 sacrificed rats' organs at different days post infection & post challenge.
Examined organs
Antigen used in
Larvae mean number at different days post infection or post challenge
immunization
3
7
15
30
45
ESAg
26
14.5
11
5.5
3
Liver
PeAg
0
0
0
0
0
EEAg
41.5
12
9
9.5
7
No. of larvae in control positive rats
998.5
328
103.5
39.5
12
No. of larvae in adjuvant group
983
311.5
89
26
8.5
ESAg
0
5
0
0
0
Lung
PeAg
0
0
0
0
0
EEAg
0
10
5.5
0
0
No. of larvae in control positive rats
0
512
126
47
16.5
No. of larvae in adjuvant group
0
523
121.5
39
10
ESAg
0
2.5
0
0
0
Spleen
PeAg
0
0
0
0
0
EEAg
0
4.5
2
0
0
No. of larvae in control positive rats
0
13
26
9.5
4
No. of larvae in adjuvant group
0
16
27.5
7
3.5
ESAg
0
0
0
0
0
Kidney
PeAg
0
0
0
0
0
EEAg
0
3
0
0
0
No. of larvae in control positive rats
0
26
14
5
0
No. of larvae in adjuvant group
0
23.5
11
3.5
0
ESAg
0
0
0
0
0
Brain
PeAg
0
0
0
0
0
EEAg
0
0
0
0
0
No. of larvae in control positive rats
0
8
12
13.5
3.5
No. of larvae in adjuvant group
0
6
11
11.5
3
ESAg
0
0
0
0
0
Muscles
PeAg
0
0
0
0
0
EEAg
0
0
2
0
0
No. of larvae in control positive rats
0
0
19
36
51
No. of larvae in adjuvant group
0
0
20
36.5
54
Table 4: Level of protection induced by T. vitulorum antigens in immunized rats at different days post challenge.
Total mean No.
% of protection after immunization with
ESAg
PeAg
EEAg
Time of
of extracted
Total mean No.
Total mean No.
slaughtering
control +ve
% of
% of
Total mean No. of
% of
of extracted
of extracted
larvae
protection
protection
extracted larvae
protection
larvae
larvae
3 dp.c.
998.5
26
97.4
0
41.5
95.8
7 dp.c.
887
22
97.5
0
29.5
96.7
15 dp.c.
300.5
11
96.4
0
100
18.5
93.8
30 dp.c.
150.5
5.5
96.4
0
9.5
93.7
45 dp.c.
87
3
96.6
0
7
95.9
Table 5: Size variations in 7 days old larvae extracted from immunized and control positive rats.
Larvae extracted from
Organs
ESAg immunized group
EEAg immunized group
Control positive group
L X D (μ)
Mean size (μ)
L X D (μ)
Mean size (μ)
L X D (μ)
Mean size (μ)
Liver
360- 440x15-18
385.7±12x16.8±0.7
362- 442x16-18
386.2±12x17.2±1.4
367-446x16-20
391.6±8x18.4±2.1
Lung
360- 459x16-20
388±10x18±1.2
362-461x16-20
389.1±9x18.5±1.5
369-461x17-21
399.8±16x18.8±2.3
Spleen
366- 468x16-20
411.3±10x18±1.8
369-462x17-20
408.6±9x18.3±1.8
371-469x18-22
413.5±11x19.6±2.6
Kidney
--
--
368- 460x17-20
414.3±9x18±2
370-468x17-22
418±18x18.2±3.1
Brain
--
--
--
--
373-468x18-22
419.±10x19.4±2.9
*L X D (μ) = Lengthx diameter (μ).
Changes in level of anti-T. vitulorum anti-
bodies (ATv-Ab) after challenge and infec-
tion of different rat groups: Immunization of
rats by the 3 tested T. vitulorum antigens
revealed high Ab titre at the end of immun-
ization period (35th dp.im.) with marked su-
periority to PeAg as the best immunogenic
type (mean OD was 0.923) followed by the
ESAg (0.862) and the lowest level of pro-
duced Ab level was in the EEAg immunized
group (0.728) as compared with the ELISA
OD value of each rat at day zero of immun-
ization trial and with adjuvant group.
The challenge of each rat with 10, 000 em-
bryonated eggs revealed gradual increase in
the level of antibodies in all of immunized
groups till the period end (45th dp.c.). The
recorded levels of antibodies in each time
still related to the primary level of antibod-
ies produced from each Ag before challenge.
298
In this respect, Ab level in the group im-
munized by PeAg still that have the highest
level of Ab in their post challenge sera. It
was noticed that the pattern of changes in
ATv-Ab titre in control positive rats differed
than that in immunized rats as it increased
gradually from 0.176 at 3rd dp.i. to 0.547 at
45 d.pi. This rate of gradual increase in con-
trol positive rats was higher than that in the
adjuvant group (Tab. 6 & Fig. 3).
Table 6: Changes in the level of ATv-Ab in immunized and control rats post challenge & infection.
Time of Ab level
Changes in Ab titter in (GA immunized by
Changes in Ab titter in controls
determination
ESAg
PeAg
EEAg
(GB)
(GC)
(GD)
Zero day
0.141
0.148
0.152
0.154
0.138
0.125
35 dp.im.
0.862
0.923
0.728
0.147
0.304
0.129
3 dp.i./p.c.
0.592
0.719
0.509
0.176
0.263
0.167
7 dp.i./ p.c.
0.634
0.782
0.593
0.288
0.281
0.181
15 dp.i/ p.c.
0.678
0.881
0.613
0.480
0.411
0.154
30 dp.i/ p.c.
0.776
0.914
0.661
0.502
0.456
0.130
45 dp.i./ p.c.
0.839
0.958
0.689
0.547
0.489
0.143
*Cut off value = 0.290
Discussion
Toxocara vitulorum infection is an im-
portant cause of losses in buffaloes and
calves whose economic importance cannot
be neglected. Moreover, it considered as a
probable cause for VLM in Egyptian people
as they are of low contact to dogs. For these
reasons the present study aimed to investi-
gate the efficacy of vaccination by different
T. vitulorum antigens as a way for protection
from infection by this parasite.
In the present study, rats’ infection with T.
vitulorum embryonated eggs revealed ap-
pearance of high number of larvae in liver at
3 dp.i. and then the larvae spread to the rest
of tissues at 7th dp.i. with high number in
the lung. While the larvae extracted from the
previouse organs and muscles at 15th d.p.i.
and still diagnose in these organs till the end
of the experiment. This migratory pattern
matched with Omar and Barriga (1991) who
reported that the larvae found in the highest
number in liver at first days p.i then spread
to the rest of tissues with decreasing num-
ber. Alsom the results agreed with Strube et
al. (2013) who described the migratory pat-
tern in different paratenic hosts and also
mentioned that T. vitulorum larvae had low
affinity to the brain.
In the present study, it was found that im-
munization by PeAg showed 100% protec-
tion versus challenge infection, followed by
the ESAg (96.4-97.5%) and EEAg (93.7-
96.7%). This reflects the migratory behavior
of T. vitulorum larvae in the immunized rats
as the number of larvae extracted from those
rats was lower than that extracted from the
control positive rats. This result agreed with
Amerasinghe et al. (1992) found that peAg
was most protective antigen against T. vitul-
orum infection in mice followed by ESAg.
El-Askalany et al. (2008) reported that im-
munization of rabbits with EEAg decreased
the tissue invasion with larvae. Omar and
Barriga (1991) explained that the decrease in
number of the extracted larvae may be due
to intestinal resistance which inhibited
hatching and prevention of larvae from mu-
cosal penetration. Even if some embryonat-
ed eggs were hatched; the larvae might be
killed by the action of tissue defense mecha-
nism (macrophages), or by the action of the
eosinophilic degranulation, Capron (1991).
While Ruppel et al. (1990) reflected the de-
crease in number due to reduction in the rate
of migration of the larvae to the tissues and
killing some of the parasites during the first
3 weeks of infection.
In the present study, PeAg showed the
most immunogenic T. vitulorum antigen ca-
using high Ab-titre at 35th dp.im. followed
by ESAg and the lowest level was in group
immunized by EEAg using ELISA. These
results were confirmed by the data obtained
after postmortem examination, as immuniza-
tion by PeAg produced the highest protec-
tion level. It was noticed that the level of
antibodies in the immunized rats' sera after
299
the challenge with T. vitulorum embryonated
eggs was higher than that in the other infect-ed
rats. High positive antibodies titer was noticed
at 15 dp.i./ p.c. and remained high during the
experimental period until day 45, which
agreed with Morales et al. (2002) who
detected an increase in the level of an-tibodies
2 weeks post T. canis infection of rabbits till
the end of the experimental study.
In the present study, there was a significant
(P≤0.05) decrease in the number of the ex-
tracted larvae from immunized rats than that
extracted from other infected rats, there was
non-significant decrease in the size of ex-
tracted larvae at 7th dp.i/ p.c. from rats in
both immunized and control groups. This
result agreed with Barriga and Omar (1992)
who reported that the size of T. vitulorum
larvae in the tissues of the infected non-
immunized rabbits increased with the time.
Moreover, Warren (1971) reported that even
the larvae survived and invaded tissues of
immunized rabbits, their growth may be re-
tained. In the same time, SEM investigation
determined absence of any morphological
alternation in the structure of larvae extract-
ed from immunized and control rats.
It was worthy to mention that the 7th dp.i/
p.c. was selected as a time for evaluating the
size of the extracted larvae aiming to inves-
tigate the effect of immunization only on the
migrating larvae, as with increasing the time
post infection, some other factors may affect
the size of the produced larvae such as mi-
gration of the larvae through un-proper way
or away from good nourished organs. So
selection of this time avoids the possibility
of the effect of other factors that may inhibit
larval development other than immune-
status of the animal.
It was noticed that T. vitulorum infection in
rats in the adjuvant group had the same pat-
tern of rats in the infected non-immunized
group throughout the study. This proved that
the reported high Ab levels and immuniza-
tion effect was produced only by the action
of the injected antigens not the adjuvant.
In the present study, effect of immuniza-
tion on the invading larvae was inspected
from two main aspects, the first one was its
effect on number of larvae succeeded to
penetrate the intestinal wall of immunized
rats, while the second one was focused on
the effect of immunization on the morpho-
logical features of the penetrating larvae
from the aspect of its size and structures.
Immunization caused significant (P≤0.05)
decrease in the number of penetrating larvae
but it did not affect the size till 7 dp.i. Be-
sides, inspection of larvae using light and
SEM examination revealed no morphologi-
cal changes were detected in the extracted
larvae from immunized rats in comparison
with that extracted from non-immunized rats
after the infection. T. vitulorum morph-olo-
gy by SEM went with Ashour et al. (1996).
Immunization of rats succeeded to build
an early line of defense on the intestinal
wall. These diminished the ability of most
invading larvae to induce successful pene-
tration to intestinal wall and arrive to gen-
eral circulation. In the same time, effect of
immunization on larvae after this appear
lower than this effect as the larvae that suc-
ceed in penetration to intestinal wall reached
to different body organs.
Conclusion
The results revealed that the migratory
pattern and vaccination trials for T. vitulo-
rum infection in rats showed close results of
that studied in other paratenic hosts. There-
fore we concluded that rats can be consid-
ered as a suitable model for performing
some investigations of T. vitulorum infec-
tion in the suspected hosts and even testing
the efficacy of some control methods rather
than treatments.
Moreover, the PeAg was considered to be
valuable immunogenic and protective anti-
gen, can used in minimize infection by T.
vitulorum between mothers and calves by
vaccination in infected farms.
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Guinea pigs and mice infected with the parasitic worms Toxocara canis developed airway inflammation and tracheal hyporesponsiveness. Preceding inflammatory cell infiltration a brief hyperreactive response occurred in guinea pigs to histaminergic receptor stimulation at 3 days past infection (p.i.) and in mice to acetylcholine receptor stimulation at 1 day p.i. Few but large eosinophilic inflammatory foci developed in guinea pigs at 14 days p.i. Mice demonstrated progressive multifocal inflammation from 7 days p.i. In addition to eosinophils mouse airways were infiltrated by lymphocytes, forming perivascular and (partial) peribronchial infiltrates in an oedematous submucosa. The inflammation had resolved in guinea pigs at 35 days p.i., the trachea turning normoreactive concurrently. The inflammation persisted in mice for greater than or equal to 3 months and likewise persisted tracheal hyporeactivity. Incubation of tracheae of non-infected mice with pulmonary inflammatory cells caused a significant decrease in cholinergic receptor responsiveness. This downward shift was prevented by 60% when a cyclooxygenase inhibitor was added to the incubation medium but not when inhibitors of lipoxygenase and superoxide formation were added, suggesting the involvement of prostaglandin E(2). This suggestion was supported by the finding of significantly increased prostaglandin E(2) concentrations in the bronchoalveolar lavage fluid at 14 and 28 days p.i. It was concluded that tracheal hyporeactivity coincided with the presence of large numbers of eosinophils in the airways of both, guinea pigs and mice and that prostaglandin E(2) involvement was conceivable.