Worm and Larval Burden, Histopathological and Ultrastructural Evaluation of T. Spiralis Vaccination Using Crude Worms And/or Larvae Antigens: Experimental Studies

Introduction
Trichinellosis is a serious parasitic zoonosis

with world wide distribution world
(1)
. The causative
nematode, Trichinella infects a wide variety of
vertebrates
(2)
. There are eleven Trichinella species, 8
of them are encapsulated in host muscle tissues and
infect only mammals as T. spiralis, T. native and T.
britovi, while the other 3 species are non encapsulated
and infect mammals, birds and reptiles as T.
pseudospirallis, T. papuae and T. zimbabwensis
(3)
.
It is primarily a disease of human and pigs. Raw or
poorly cooked pork (usually sausage) harboring the
infective larvae is the common vehicle for human
infections
(4)
.
T. spiralis is considered as both an intestinal
and tissue parasite. There are two main phases
of infection; enteral (affecting the intestine)
and parenteral (outside the intestine)
(5)
. In man,
the intestinal phase presents as gastroenteritis
simulating food poisoning as early as 24 hours
after ingestion of infected pork. Larval migration
and penetration of muscles are manifested by fever,
eyelids edema, myositis and weakness of involved
Worm and Larval Burden, Histopathological and Ultrastructural Evaluation
of T. spiralis Vaccination Using Crude Worms and/or Larvae Antigens:
Experimental Studies
Nashaat E. Nassef, Mona M. El-Sobky and Amira F. Afifi
Parasitology Department, Faculty of Medicine, Menoufiya University, Egypt
27
Abstract
Background: Currently, there is no vaccine for T. spiralis; however, several studies have been made towards
understanding the immune mechanisms that contribute to host protection against it.
Objective: The aim of the present study is to evaluate the protective effect of vaccination by T. spiralis
adult, larval and combined adult and larval crude antigens against trichinellosis in experimental mice.
Methodology: Swiss male albino mice (No. = 125) were divided into 5 groups. Groups A, B and C were
immunized by T. spiralis crude larval antigen, crude worm antigen, and combined larval and worm antigens,
respectively. One week after the last dose of injection, each mouse was infected orally with 150-200 larvae.
Two other groups (D and E) served as infected non immunized control groups. Group E received adjuvant
and phosphate buffer saline before infection. At the 8th day post-infection (PI), 12 mice from each group
were sacriced and the intestinal wormburden was assessed, while the muscle larval burden was evaluated
at 28th day PI in the remaining mice of each group. Intestinal and skeletal muscle specimens were prepared
for histopathological study. Meanwhile, adults and larvae were examined by scanning electron microscope
(SEM) and infected muscle sections were examined by transmission electron microscope (TEM).
Results: Combined antigen gave the highest reduction% in intestinal worm and larval muscle burdens (92%
and 96%, respectively), followed by larval antigen (86% and 91%), then worm antigen (73% and 88%),
compared with infected non immunized control groups. Compared with groups (A and B), group C gave
signicant reduction in both intestinal and muscle burdens. Histopathological examination revealed marked
decrease in intestinal inammatory inltrates, and marked reduction of encysted larvae with mild inltration
around the degenerated larvae in mice of group C. SEM and TEM results conrmed the signicant effect
of the combined vaccine (Group C).
Conclusion: Vaccination with combined worms and larval antigens gave the most protective action against
T. spiralis challenge infection.
Recommendations: The use of combined antigen in mass vaccination of reservoir animals may decrease
the risk of human infection.
Keywords: T. spiralis, Vaccination, Crude Worm Antigen, Crude Larval Antigen, Combined Antigen, SEM, TEM.
Received: March, 2010 Accepted: May, 2010
Parasitologists United Journal (PUJ) Vol. 3, No. 1 & 2 , 2010
27 - 38
28 Nassef et al.,
muscles. There may also be a maculo-papular skin
rash, and symptoms of pneumonitis occurring
between the 2nd and 6th day post infection (PI),
lasting for 5 days. High eosinophilia from 10% to
less than 90% is pathognomonic in the 3rd or 4th
week (PI)
(6)
. Diagnosis of trichinellosis is suspected
on the basis of both epidemiological and clinical
data. Diagnosis of enteric phase must be early by
stool examination in the 1st week to detect adults
especially males and larvae. At 3rd or 4th week, the
direct diagnostic method is muscle biopsy to detect
the encysted larvae in muscles
(7)
.
Trichinella spiralis antigens either on the surface
or excreted-secreted (E/S) are key modulators
or targets of the host immune system
(8)
. Immune
responses are often intense and involve components
of humoral and cellular immune systems regulated
by T helper lymphocytes (both Th1 and Th2)
(9)
.
Activation of GIT mucosal immune system in
trichinellosis results in altered intestinal physiology,
which includes goblet cell hyperplasia with
increased mucin secretion, mast cell hyperplasia,
increase of both fluid secretion and intestinal muscle
contractility leading to worm expulsion
(10,11)
. These
are associated with marked changes in epithelial
cells, with increase in number of inflammatory cell
types and the release of inflammatory mediators
(12)
.
Currently, there are no vaccines for T. spiralis.
However, several mice studies aiming to produce
vaccine candidates have yielded promising
results
(13)
. Protective immunity against trichinellosis
has been produced by immunization with different
antigenic preparations including cuticular, E/S and
somatic antigens
(14,15)
, in addition to DNA vaccine
or recombinant antigen protein
(1,16)
.
The aim of the present work was to evaluate the
protective effect of vaccination using T. spiralis
antigens (larva, adult and combined) in experimental
mice by parasitological, histopathological and
ultrastructural studies.
Materials and Methods
Experimental animals: Male albino rats (180-220
g) were used for isolation of infection from infected pig,
maintenance of the laboratory strain, and preparation of
crude antigens, while Swiss male albino mice (18-20 g)
were used for the immunization studies.
Methods
Maintenance of T. spiralis infection in laboratory
rats: Muscle samples (diaphragm and other skeletal
muscles) of T. spiralis-infected pigs were obtained from
Cairo Slaughter House. The infected muscle samples
were selected after routine examination and isolation of
infected pigs, carried out in the slaughter house using the
trichinoscope. For propagation of T. spiralis infection, 20
rats were infected from artificially digested infected pig
muscles
(17)
. Rats were infected orally with 1 ml of larval
suspension (800-1000 larvae/rat). After 5 weeks PI, rats
were sacrificed and larvae were isolated and collected.
The excysted larvae were washed and left for 30 min for
sedimentation
(18)
. The sedimented larvae were counted
using master counting chamber. Concentrated volume of
counted larvae was adjusted so that the desired number
of larvae in a certain volume of fluid was obtained.
Antigens preparation: Twenty rats were infected
with 8000-10000 larvae/rat and sacrificed 3 days PI.
They were starved the night before sacrifice by ether. To
prepare crude adult antigen, the abdomen was dissected
from the mesentery, slit opened longitudinally, cut into
parts (5 cm each) and put in a beaker containing PBS
(pH 7.2-7.4). The beaker was rinsed 2-3 times with PBS
and its content was sieved first through a 60 meshes/cm
2
sieve to remove food particles and intestinal tissues,
followed by a 250 meshes/cm
2
sieve. Collected worms
were washed 3-5 times with PBS and homogenized
in an ultrasound tissue homogenizer, followed by
centrifugation in a cooling centrifuge at 10C, and at a
speed of 10000 rpm for one hour. The supernatant was
collected as crude adult antigen
(19)
. Another 20 rats were
infected and sacrificed 35 days PI, and muscle larvae
were obtained as previously mentioned, concentrated,
washed (3-5 times) by PBS (pH 7.2), and re-suspended in
the same fluid
(20)
. Crude antigen preparation from larvae
29
Vaccination of T. spiralis
followed the same steps for worm antigen preparation.
Protein concentration of both antigens was determined
(21)
,
then the crude antigens were collected in one ml vials and
frozen at 20C till use.
Animal vaccination (Study design): Mice (No.
= 125) were divided into 5 groups (25 mice each). In
group A, each mouse was immunized by intra-peritoneal
injection of 200 g (100 g of larval antigen mixed with
equal volume of complete Freunds adjuvant (CFA)
on the 1st day, followed by two booster doses of 50
g of antigen mixed with equal volume of incomplete
Freunds adjuvant (IFA) on days 15 and 28
(22)
. One week
after the last dose of injection, each mouse was infected
orally with 150-200 larvae
(18)
. Similar vaccination and
challenge infection schedule with crude worm antigen
were conducted in mice of group B. In group C, mice
were immunized with 200 g of combined adult and
larval antigens (100 g each) given in three doses (with
CFA in the first dose, and IFA in the booster doses).
On the other hand, mice of groups (D and E) served as
infected non immunized control groups. Group D was
infected without vaccination, while group E received
only CFA and IFA with phosphate buffer saline (PBS)
before oral infection with the same dose and procedure
as in vaccinated groups.
Effects of the used vaccines: At day 8 PI, 12 mice
from each group were sacrificed, and the intestinal
worm burden was measured
(23)
, while the muscle larval
burden was evaluated
(24)
in the remaining mice on day
28 PI. For the histopathological studies, 1 cm from the
intestine at the junction of proximal 1/3 and distal 2/3
were taken on day 8 PI and specimens from the skeletal
muscles of the hind limbs were also taken on day 28 PI.
All specimens were fixed in 10% formalin, dehydrated
in ascending grades of ethyl alcohol and cleared in
xylol, then embedded in paraffin
(25)
. Sections about 3-4
thick were cut by microtome and prepared for staining
by hematoxylin and eosin
(26)
. The parasitological and
pathological studies were conducted in Parasitology and
Pathology Departments, Faculty of Medicine, Menoufiya
University. On the other hand, ultrastructural studies
(27)
,
including SEM for T. spiralis worms and larvae, and TEM
for infected muscles were done in Anatomy Department,
Faculty of Medicine, Ain Shams University, and Electron
Microscopic Unit, Faculty of Science, Alexandria
University, respectively.
Statistical analysis: Results were collected, tabulated
and statistically analyzed using statistical package SPSS
(16.0). The reduction % was calculated in the vaccinated
groups in comparison with group D. The means of the
different groups were compared globally using the
analysis of variance (ANOVA) and between groups using
the student t test. The results were considered significant
if P < 0.05
(28)
.
Ethical consideration: The experimental animal
studies were conducted in accordance with the
international valid guidelines and they were maintained
under convenient conditions at the animal house in
Pharmacology Department, Faculty of Medicine,
Menoufiya University.
Results
Global statistical analysis using ANOVA showed
significant reduction in the mean number of adult
worms (86%, 73% and 92, respectively) in all
vaccinated groups compared with non immunized
infected control groups (P<0.0001). No significant
difference was found between infected control and
infected adjuvant groups (D and E) (P>0.05) (Table
1). Using student t test, there was significant reduction
of worms in group C compared with groups A and
B (P<0.001 and P<0.001, respectively). In addition,
mice of group A showed significant reduction of
worms compared with group B (P<0.001) (Table
2). Similar results were obtained regarding global
statistical analysis using ANOVA in the mean larval
count in the three vaccinated groups (91%, 88%
and 96% reduction, respectively) (Table 1). On
the other hand, significant reduction in the mean
larval count (P<0.001 and P<0.001) was obtained
in comparison between groups (C and A) and (C
and B), respectively. The results were significant
(P<0.05) in comparison between the mean larval
count of mice of groups (A and B) (Table 2).
Results of the histopathological studies are
illustrated in figures (1-8), SEM results in figures
(9-16), and TEM results in figures (17-24).
Histopathological examination of intestinal sections
of vaccinated groups (Figures 2-4) compared to the
infected control group D (Figure 1) revealed very
30 Nassef et al.,
marked decrease in inflammatory infiltrates in group
C, while mild to moderate intestinal inflammations
were observed in groups A and B. Compared to group
D (Figure 5), muscle sections showed reduction of
encysted larvae with mild surrounding infiltration,
and hyaline degeneration of the larvae. In addition,
there was mild muscle degeneration adjacent to the
cyst with normal distant muscle. These findings
were obvious in muscles recovered from mice of
group C (Figure 6) and varied from mild to moderate
in groups A and B (Figures 7 and 8).
Examination of worms and larvae by SEM
revealed that with combined antigen (Figures 11
and 12), there was marked destruction of larvae and
worms with marked swellings, disintegration of the
cuticles, multiple large blebs and vesicles with loss
of normal morphology and architecture of the cuticle
compared to infected control group (D) (Figures 9
and 10). Larval and worms antigens gave the same
results but with lower degree of destruction (Figures
13-16). TEM of muscle sections showed mild to
moderate enlargement and increase in number of
mitochondria with moderate depletion of glycogen,
slight disturbance of Z line, intact myofillaments
with normal nucleus and mild to moderate dilatation
of endoplasmic reticulum. These findings were more
obvious with combined antigen (Figures 19 and 20)
than the other antigens (Figures 21-24) compared to
normal and infected control groups D and E (Figures
17 and 18).
B) (Table 2).
Results oI the histopathological studies are illustrated in Iigures (1-8), SEM results in Iigures
(9-16), and TEM results in Iigures (17-24). Histopathological examination oI intestinal
sections oI vaccinated groups (Figures 2-4) compared to the inIected control group D (Figure
1) revealed very marked decrease in inIlammatory inIiltrates in group C, while mild to
moderate intestinal inIlammations were observed in groups A and B. Compared to group D
(Figure 5), muscle sections showed reduction oI encysted larvae with mild surrounding
inIiltration, and hyaline degeneration oI the larvae. In addition, there was mild muscle
degeneration adjacent to the cyst with normal distant muscle. These Iindings were obvious in
muscles recovered Irom mice oI group C (Figure 6) and varied Irom mild to moderate in
groups A and B (Figures 7 and 8).
Examination oI worms and larvae by SEM revealed that with combined antigen (Figures 11
and 12), there was marked destruction oI larvae and worms with marked swellings,
disintegration oI the cuticles, multiple large blebs and vesicles with loss oI normal
morphology and architecture oI the cuticle compared to inIected control group (D) (Figures 9
and 10). Larval and worms antigens gave the same results but with lower degree oI
destruction (Figures 13-16). TEM oI muscle sections showed mild to moderate enlargement
and increase in number oI mitochondria with moderate depletion oI glycogen, slight
disturbance oI Z line, intact myoIillaments with normal nucleus and mild to moderate
dilatation oI endoplasmic reticulum. These Iindings were more obvious with combined
antigen (Figures 19 and 20) than the other antigens (Figures 21-24) compared to normal and
inIected control groups D and E (Figures 17 and 18).

Table (1): Global statistical analysis using ANOVA in the studied groups
Studied Groups Intestinal Worm Count Muscle Larval Count
Mean SD
(Range)
Reduction Mean SD
(Range)
Reduction
Group A (Larval antigen) 9.77 + 2.2
(0-18)
86 1887.6 + 528.01
(950-2825)
91
Group B (Adult antigen) 19.33 + 3.8
(8-30)
73 2440.36 + 509.1
(1000-3880)
88
Group C (Combined antigen) 5.45 + 1.8
(0-10)
92 868.1 + 450.2
(390-1346)
96
Group D (Non-immunized) 71.2 + 3.6
(62-79)
0 20325.4 +
2092.3
(18215-23573)
0
Group E (Adjuvant immunized) 67.9 + 8.4
(50-80)
0 19338.6 +
2222.6
(16018-21207)
0
Global statistical analysis ANOVA test 80.8
0.0001 (SigniIicant)
ANOVA test 175.8
Individual statistical analysis
Between groups D and E
test 1.29
0.21 (Non signiIicant)
test 1.17

Table (2): Individual statistical analysis using -test in the vaccinated groups
Groups (A) vs (B) Groups (A) vs (C) Groups (B) vs (C)
Intestinal Worm Count

Muscle Larval Count
test 7.52
0.001
test 2.72
0.05
test 5.26
0.001
test 5.3
0.001
test 11.41
0.001
test 8.34
0.001
B) (Table 2).
Results oI the histopathological studies are illustrated in Iigures (1-8), SEM results in Iigures
(9-16), and TEM results in Iigures (17-24). Histopathological examination oI intestinal
sections oI vaccinated groups (Figures 2-4) compared to the inIected control group D (Figure
1) revealed very marked decrease in inIlammatory inIiltrates in group C, while mild to
moderate intestinal inIlammations were observed in groups A and B. Compared to group D
(Figure 5), muscle sections showed reduction oI encysted larvae with mild surrounding
inIiltration, and hyaline degeneration oI the larvae. In addition, there was mild muscle
degeneration adjacent to the cyst with normal distant muscle. These Iindings were obvious in
muscles recovered Irom mice oI group C (Figure 6) and varied Irom mild to moderate in
groups A and B (Figures 7 and 8).
Examination oI worms and larvae by SEM revealed that with combined antigen (Figures 11
and 12), there was marked destruction oI larvae and worms with marked swellings,
disintegration oI the cuticles, multiple large blebs and vesicles with loss oI normal
morphology and architecture oI the cuticle compared to inIected control group (D) (Figures 9
and 10). Larval and worms antigens gave the same results but with lower degree oI
destruction (Figures 13-16). TEM oI muscle sections showed mild to moderate enlargement
and increase in number oI mitochondria with moderate depletion oI glycogen, slight
disturbance oI Z line, intact myoIillaments with normal nucleus and mild to moderate
dilatation oI endoplasmic reticulum. These Iindings were more obvious with combined
antigen (Figures 19 and 20) than the other antigens (Figures 21-24) compared to normal and
inIected control groups D and E (Figures 17 and 18).

Studied Groups Intestinal Worm Count Muscle Larval Count
Mean SD
(Range)
Reduction Mean SD
(Range)
Reduction
Group A (Larval antigen) 9.77 + 2.2
(0-18)
86 1887.6 + 528.01
(950-2825)
91
Group B (Adult antigen) 19.33 + 3.8
(8-30)
73 2440.36 + 509.1
(1000-3880)
88
Group C (Combined antigen) 5.45 + 1.8
(0-10)
92 868.1 + 450.2
(390-1346)
96
Group D (Non-immunized) 71.2 + 3.6
(62-79)
0 20325.4 +
2092.3
(18215-23573)
0
Group E (Adjuvant immunized) 67.9 + 8.4
(50-80)
0 19338.6 +
2222.6
(16018-21207)
0
Global statistical analysis ANOVA test 80.8
ANOVA test 175.8
Individual statistical analysis
Between groups D and E
test 1.29
test 1.17

Table (2): Individual statistical analysis using -test in the vaccinated groups
Groups (A) vs (B) Groups (A) vs (C) Groups (B) vs (C)
Intestinal Worm Count

Muscle Larval Count
test 7.52
0.001
test 2.72
0.05
test 5.26
0.001
test 5.3
0.001
test 11.41
0.001
test 8.34
0.001
Table (2): Individual statistical analysis using t-test in the vaccinated groups
31

(1) (2) (3)

(4) (5) (6)

(7) (8)
Figure (1): H & E transverse intestinal section oI inIected control mouse showing severe inIlammatory inIiltrates in
the submucosa and the core oI atrophied villi with cut section oI parasite (P) between the villi and in intestinal lumen.
Figure (2): Intestinal section oI mouse vaccinated with combined antigen showing mild inIlammatory inIiltrates with
apparently intact mucosa and villi.
Figure (3): Intestinal section oI mouse vaccinated with crude larval antigen showing moderate inIlammatory
inIiltrates in the submucosa and the core oI villi with less atrophy oI villi.
Figure (4): Intestinal section oI mouse vaccinated with crude adult antigen showing moderate to severe inIlammatory
inIiltrates in the submucosa and in the core oI the villi with ulceration (U) and necrosis (N).
Figure (5): Skeletal muscle section oI inIected control mouse showing multiple larval depositions with severe
inIlammatory inIiltrates and degeneration oI muscle Iibers.
Figure (6): Skeletal muscle section oI mouse vaccinated with combined antigen showing very small one larval cyst
(C) with degenerated capsule, eosinophilic material replaced the larvae, mild pre-larval inIiltrates and no
degenerative changes in ms Iibers.
Figure (7): Skeletal muscle section oI mouse vaccinated with larval antigen showing multiple larval cyst with thin
capsule, mild to moderate pre-cyst inIiltration (I), eosinophilic material replaced the larvae (E) and minimal
degeneration oI muscle Iibers.
Figure (8): Skeletal muscle section oI mouse vaccinated with adult antigen showing multiple larval deposition with
moderate cellular inIiltration around thick capsule (TC) and moderate to severe degeneration oI ms Iibers with
moderate separation oI ms Iibers.

Figure (4): Intestinal section of mouse
vaccinated with crude adult
antigen showing moderate to
severe inflammatory infiltrates in
the submucosa and in the core of
the villi with ulceration (U) and
necrosis (N).
Figure (5): Skeletal muscle section of
infected control mouse showing
multiple larval depositions with
severe inflammatory infiltrates and
degeneration of muscle fibers.
Figure (6): Skeletal muscle section of mouse
vaccinated with combined antigen
showing very small one larval
cyst (C) with degenerated capsule,
eosinophilic material replaced the
larvae, mild pre-larval infiltrates
and no degenerative changes in ms
fibers.
vaccinated with adult antigen showing
multiple larval deposition with moderate
cellular infiltration around thick capsule
(TC) and moderate to severe degeneration
of ms fibers with moderate separation of
ms fibers.

(1) (2) (3)

(4) (5) (6)

(7) (8)

(1) (2) (3)

(4) (5) (6)

(7) (8)

vaccinated with larval antigen showing
multiple larval cyst with thin capsule,
mild to moderate pre-cyst infiltration
(I), eosinophilic material replaced the
larvae (E) and minimal degeneration of
muscle fibers.

(1) (2) (3)

(4) (5) (6)

(7) (8)

Figure (1): H & E transverse intestinal
section of infected control mouse
showing severe inflammatory
infiltrates in the submucosa and
the core of atrophied villi with cut
section of parasite (P) between the
villi and in intestinal lumen.
showing mild inflammatory
infiltrates with apparently intact
mucosa and villi.
vaccinated with crude larval
antigen showing moderate
inflammatory infiltrates in the
submucosa and the core of villi
with less atrophy of villi.
32 Nassef et al.,
Figure (16): SEM of T. spiralis larva of mouse
moderate oedema that cause obliteration
of the longitudinal furrow, moderate loss
of folds, ridges and multiple small blebs.
Figure (15): SEM of T. spiralis adult of mouse
multiple blebs (B), irregular configuration
of the cuticle and loss of ridges and folds.

(9) (10) (11)

(12) (13) (14)

(15) (16)
Figure (9): SEM oI T. spiralis adult oI an inIected control mouse showing cephalic structure oI
anterior-lateral papillae (P) Iound on broad bulk showing primary Iolds with large spacing. Fine
longitudinal ridges (L), transverse creases (C) and hypodermal glands opening (G) are seen.
Figure (10): SEM oI T. spiralis larva oI an inIected control mouse showing oral slit (O.S.) like
opening which slightly depressed below the surIace, transverse Iolds with narrow spacing in
between and longitudinal Iurrow with no opening or pores on the lateral surIace.
Figure (11): SEM oI T. spiralis adult oI mouse vaccinated with combined antigen showing
multiple Iissures (F), multiple blebs (B) some oI them are ruptured and complete destruction oI the
cuticle.
Figure (12): SEM oI T. spiralis larva oI mouse vaccinated with combined antigen showing severe
edematous cuticle with Iormation oI large vesicles (V) and multiple blebs with loss oI normal
Ieatures oI the cuticle.
Figure (13): SEM oI T. spiralis adult oI mouse vaccinated with larval antigen showing severe
sloughing oI cuticle with loss oI some area, severe oedema with large Iissuring (F).
Figure (14): SEM oI T. spiralis larva oI mouse vaccinated with larval antigen showing large
swelling (S), moderate oedema, multiple blebs (B) with area oI Iissuring (F) and some cuticlular
sloughing.
Figure (15): SEM oI T. spiralis adult oI mouse vaccinated with adult antigen showing multiple
blebs (B), irregular conIiguration oI the cuticle and loss oI ridges and Iolds.
Figure (16): SEM oI T. spiralis larva oI mouse vaccinated with adult antigen showing moderate
oedema that cause obliteration oI the longitudinal Iurrow, moderate loss oI Iolds, ridges and
multiple small blebs.

(9) (10) (11)

(12) (13) (14)

(15) (16)
cuticle.
sloughing.

(9) (10) (11)

(12) (13) (14)

(15) (16)
cuticle.
sloughing.

(9) (10) (11)

(12) (13) (14)

(15) (16)
cuticle.
sloughing.

(9) (10) (11)

(12) (13) (14)

(15) (16)
cuticle.
sloughing.

Figure (9): SEM of T. spiralis adult of an
infected control mouse showing
cephalic structure of anterior-lateral
papillae (P) found on broad bulk
showing primary folds with large
spacing. Fine longitudinal ridges (L),
transverse creases (C) and hypodermal
glands opening (G) are seen.
Figure (10): SEM of T. spiralis larva of
an infected control mouse showing
oral slit (O.S.) like opening which
slightly depressed below the surface,
transverse folds with narrow spacing
in between and longitudinal furrow
with no opening or pores on the
lateral surface.
Figure (11): SEM of T. spiralis adult of
mouse vaccinated with combined
antigen showing multiple fissures
(F), multiple blebs (B) some of
them are ruptured and complete
destruction of the cuticle.
Figure (13): SEM of T. spiralis adult
of mouse vaccinated with larval
antigen showing severe sloughing
of cuticle with loss of some area,
severe oedema with large fissuring
(F).
Figure (14): SEM of T. spiralis larva
antigen showing large swelling
(S), moderate oedema, multiple
blebs (B) with area of fissuring (F)
and some cuticlular sloughing.

(9) (10) (11)

(12) (13) (14)

(15) (16)
cuticle.
sloughing.

(9) (10) (11)

(12) (13) (14)

(15) (16)
cuticle.
sloughing.

(9) (10) (11)

(12) (13) (14)

(15) (16)
cuticle.
sloughing.

Figure (12): SEM of T. spiralis larva of mouse
showing severe edematous cuticle
with formation of large vesicles (V)
and multiple blebs with loss of normal
features of the cuticle.
33
Figure (24): TEM of skeletal muscle
of mouse vaccinated with adult
antigen showing degeneration of
myofillament (D.Mf) with enlarged
mitochondria (M) that increased in
number, disturbance of Z. line and
marked depletion of glycogen (G).

(17) (18) (19)

(20) (21) (22)

(23) (24)
Figure (17): TEM oI skeletal muscle oI normal control mouse showing normal muscle Iibers, normal glycogen
distribution (G), good appearance oI Z. line, average size, number and distribution oI mitochondria (M) and
normal appearance oI smooth endoplasmic reticulum (SER).
Figure (18): TEM oI skeletal muscle oI inIected control mouse showing encysted larva (L) with highly inIiltrated
capsule (C) by polymorphonuclear cells (P). Muscle surrounding showing highly degeneration, loss oI striation
and Z. line, loss oI glycogen and abnormal shape oI muscle cell nucleus (N) with irregular distribution oI
chromatin materials.
Figure (19): TEM oI skeletal muscle oI mouse vaccinated with combined antigen showing slight dilatation oI
mitochondria (M) with average number, average glycogen (G), normal Z. line, good striation oI myoIillaments,
mild to moderate vacculation (V) and average size and number oI S.E.R.
Figure (20): TEM oI skeletal muscle oI mouse vaccinated with combined antigen showing normal nucleus oI
muscle cell, average glycogen (G), average number and size oI mitochondria (M), interrupted Z. line and slight
vacculation (V).
Figure (21): TEM oI skeletal muscle oI mouse vaccinated with larval antigen showing marked dilatation oI
S.E.R., average glycogen (G), disturbance oI Z. line, mild dilatation and rupture oI some mitochondria (M) and
moderate vacculation.
Figure (22): TEM oI skeletal muscle oI mouse vaccinated with larval antigen showing thick collagen capsule (C)
oI encysted larva inIiltrated with polymorphonuclear leukocytes (P) and the muscle (Ms) adjacent showing no
degeneration.
Figure (23): TEM oI skeletal muscle oI mouse vaccinated with adult antigen showing nurse cell oI the parasite
including part oI the capsule (C) inIiltrated with large number oI plymorphonuclear cells (P.C) mostly Iibroblast
and macrophage surrounding a part oI the larva (L). Part oI muscle cells (Ms) showing normal appearance.
Figure (24): TEM oI skeletal muscle oI mouse vaccinated with adult antigen showing degeneration oI
myoIillament (D.MI) with enlarged mitochondria (M) that increased in number, disturbance oI Z. line and
marked depletion oI glycogen (G).

(17) (18) (19)

(20) (21) (22)

(23) (24)
vacculation (V).
degeneration.

(17) (18) (19)

(20) (21) (22)

(23) (24)
vacculation (V).
degeneration.

Figure (22): TEM of skeletal muscle
antigen showing thick collagen
capsule (C) of encysted larva
infiltrated with polymorphonuclear
leukocytes (P) and the muscle (Ms)
adjacent showing no degeneration.
Figure (23): TEM of skeletal muscle of mouse
nurse cell of the parasite including
part of the capsule (C) infiltrated with
large number of plymorphonuclear
cells (P.C) mostly fibroblast and
macrophage surrounding a part of the
larva (L). Part of muscle cells (Ms)
showing normal appearance.

(17) (18) (19)

(20) (21) (22)

(23) (24)
vacculation (V).
degeneration.

(17) (18) (19)

(20) (21) (22)

(23) (24)
vacculation (V).
degeneration.

Figure (19): TEM of skeletal muscle of
antigen showing slight dilatation
of mitochondria (M) with average
number, average glycogen (G),
normal Z. line, good striation of
myofillaments, mild to moderate
vacculation (V) and average size
and number of S.E.R.
antigen showing normal nucleus
of muscle cell, average glycogen
(G), average number and size of
mitochondria (M), interrupted Z.
line and slight vacculation (V).
mouse vaccinated with larval antigen
showing marked dilatation of S.E.R.,
average glycogen (G), disturbance
of Z. line, mild dilatation and rupture
of some mitochondria (M) and

(17) (18) (19)

(20) (21) (22)

(23) (24)
vacculation (V).
degeneration.

(17) (18) (19)

(20) (21) (22)

(23) (24)
vacculation (V).
degeneration.

(17) (18) (19)

(20) (21) (22)

(23) (24)
vacculation (V).
degeneration.

normal control mouse showing normal
muscle fibers, normal glycogen
distribution (G), good appearance
of Z. line, average size, number and
distribution of mitochondria (M)
and normal appearance of smooth
endoplasmic reticulum (SER).
Figure (18): TEM of skeletal muscle of infected
control mouse showing encysted larva
(L) with highly infiltrated capsule
(C) by polymorphonuclear cells (P).
Muscle surrounding showing highly
degeneration, loss of striation and Z.
line, loss of glycogen and abnormal
shape of muscle cell nucleus (N) with
irregular distribution of chromatin
materials.
34 Nassef et al.,
Discussion
Major advances have been made in the past
several years towards understanding the immune
mechanisms that contribute to host protection
against intestinal nematode parasites. These
insights may prove useful in the development of
new immunologically based treatments including
vaccines to enhance resistance to intestinal nematode
parasites
(29)
. Several researches have been done to
modify antigens derived from T. spiralis in order to
increase their specificity and subsequently increase
their efficacy as vaccines
(30)
. Moreover, the history
of protective antigens of T. spiralis suggests that the
prospect of developing vaccines for trichinellosis is
promising
(31)
.
In the present study, intestinal worm burden
of all vaccinated groups was evaluated. Results
showed significant reduction of 86%, 73% and 92%
in worm burden in groups A (larval), B (adult) and
C (combined), respectively compared to group D
(P<0.0001). Other studies
(14,32,33)
reported reduction
of worm burden of 70.9%,74% and 82%, respectively
on using crude larval antigen. On the other hand,
higher percentage reduction in worm burden was
obtained (98.4% and 94.5%) using E/S antigen of
larval stage without adjuvant
(22)
, and autoclaved T.
spiralis larval vaccine in combination with bacille
Calmette Guerin (BCG) as an adjuvant
(31)
. The use of
a copolymer microcapsule containing first stage T.
spiralis larval antigen to escape the effect of gastric
acids on the larvae
(13)
, increased CD4+ cells and
antigen-specific serum IgG and IgA. This resulted
in a statistically significant reduction (P<0.05) in
the intestinal worm burden.
Regarding the use of adult antigen for
vaccination, a higher percentage reduction in worm
burden (89% compared to 73% in the present
study) was reported
(32)
. Recently, it was shown that
while both worm and muscle larval antigens of T.
spiralis could stimulate mice to produce protective
immunity, mice immunized with worm antigen were
more resistant to subsequent challenge infection
leading to significant reduction in worm and larval
burdens
(15)
.
In the present study, results of the effect of the
three varieties of vaccines revealed that combined
antigen (Group C) showed the highest reduction
rate in worm burden and gave a potent host
resistant immunity against challenge infection with
T. spiralis compared with infected control group
(P<0.0001). Likewise, Eissa et al.
(34)
found that
the mixed components of crude adult and larval
antigens caused significant reduction of 93% in
worm burden.
Reduction in worm burden in all vaccinated
groups may be attributed to activation of the
host immunity against the intestinal phase of the
parasite through changes in gut physiology, which
is accompanied by mucosal mast cell and goblet
cell hyperplasia with release of their mediators in
the gut lumen
(35
). Both mast cell and goblet cell
hyperplasia are highly dependant on mucosal T
cells, more specifically on Th2 cytokines including
interleukins (IL- 4 and 13)
(11)
. Recently, it was
reported that mucosal mast cell mediators including
histamine, cytokines and serine proteases, such as
mast cell protease-1 (Mcpt-1), have a role in the
regulation of inflammation and tissue remodeling
as well as increasing epithelial permeability
(29)
. On
the other hand, mucus released by goblet cells acts
as a protective barrier to intestinal epithelium and
effector molecules such as intelectin and resistin
like molecule (RELM ). The latter may function
by contributing to nematode expulsion by altering
mucus composition and may also have antimicrobial
function
(35)
. Regarding, the humoral response, it was
found that neither B cells nor specific antibody are
required for T. spiralis expulsion from the intestine
(36)
.
However, there is some support for the role of parasite
specific IgE in both the intestinal response to adult
T. spiralis and larvae in the tissues
(37)
. In addition,
intestinal smooth muscle hypercontractility, under
the influence of Th2 immune response and their
cytokines, was reported
(38)
to affect the secondary
messenger system of muscle cells. This was said to
occur by acting either on the calcium channels or on
the muscarinic receptor, which subsequently alters
the intestinal muscle contractility.
Muscle larval burden of vaccinated groups was
evaluated, and results showed significant reduction
of muscle larval burden (91%, 80% and 96%, with
P < 0.0001) for groups A, B and C, respectively,
35
compared with the infected control group D. Other
reports
(31,32)
confirmed that immunization of mice
using autoclaved T. spiralis larval vaccine with
BCG (adjuvant) and crude larval antigen caused
reduction of 94.4% and 89.5%, respectively in
muscle larval burden. On the other hand, lower
percentage reduction in muscle larval burden was
reported in several studies using larval antigen
(88%
(14)
, 82.9%
(22)
, 82%
(33)
and 79%
(39)
, compared
to 91% in the present study). It was reported
that oral vaccination with the copolymer micro-
encapsulated crude larval extracts and E/S products
induced stimulation of IF- secretion and inhibition
of IL-4 secretion in spleen and mesenteric lymph
nodes of immunized mice
(13)
. This method induced
concurrent Th1/ Th2 local and systemic responses
that are protective, and at the same time may help
balancing the strong Th2 response triggered by
helminth infections. Immunization of mice with
DNA vaccine induced both humoral and cellular
immune responses which provided partial protection
against challenge infection with T. spiralis, as shown
by significant reduction of muscle larval burden
(1)
.
Compared with our results, 80% reduction in
muscle larval burden was reported by others
(32)
using
crude adult antigen, while another study reported that
mice immunized with worm antigen produced more
resistance to the subsequent challenge infection
leading to significant reduction in worm and muscle
larval burden
(15)
. Significantly the combined antigen
produced higher reduction in muscle larvae (96%)
in group C than the other antigens in groups A and
B (P<0.001). Meanwhile, Eissa et al.
(34)
showed that
combined worm and larval antigen gave a reduction
rate of 90.5% in muscle larval burden.
The highest reduction in muscle larvae with
combined antigen vaccination may be attributed
to the development of mice protective immunity
against T. spiralis infection. It was reported that
T. spiralis larval antigen in addition to worm
antigen are highly immunogenic and have a role
in the induction of innate immune responses with
particular emphasis on the activation of mast cells
and the released mediators which have a role in
the development of type 2 immune response
(40)
. In
addition to stimulation of antibody responses (IgG1,
IgG2 and IgE) against migrating newborn larvae
and chronic muscle infection
(9)
, cellular infiltration
was reported to occur at the site of infected muscle
where macrophages were the predominant cells,
but also included numerous CD4 T cells, fewer
CD8 T cells and rare B lymphocytes
(41,42)
. Despite a
potent and persistent B cell response during muscle
infection, the local inflammatory response to the
T. spiralis nurse cell is limited, suggesting that
suppressive parasite or host factors are functioning.
It was observed that the infected muscle remodels
the cytoplasm matrix, synthesizes a collagen and
induces the formation of alterations in host gene
expression
(43)
. Transcription of muscle-specific
genes falls dramatically
(44)
, while synthesis of
syndecan-1 is induced
(41)
, vascular endothelial
growth factor genes are activated
(45)
and collagen
transcripts are increased
(46)
. These changes correlate
with the formation of the nurse cell
(43)
. Moreover,
resistance of the infective stage of the parasite in
muscle tissue is partially dependent on CD4 T cell-
regulated levels of IL-10 and transforming growth
factor beta (TGF-) that normally control the level
of inflammation surrounding the parasite-modified
nurse cell during the early stages of muscle infection
(20 days PI). While control of chronic inflammation
between 20 and 50 days PI is IL-10 independent and
is coincident with the emergence of a potent Th2
response
(42,43)
.
Histopathological results in the present study
showed decrease in the inflammatory response
for T. spiralis infection in intestinal and muscle
sections with reduction of encysted muscle larvae of
all vaccinated groups compared to infected control
group. These changes were obvious with combined
antigen than other antigens. Another study
(31)
reported reduction in encysted muscle larvae with
mild inflammatory infiltrate by using autoclaved T.
spiralis larval antigen in combination with BCG.
Results of SEM and TEM revealed that combined
antigen produced more destruction to worms and
larvae with more improvement of muscle cells
than other antigens. The effect of vaccination
might be due to increase of IgG antibody in the
sera of immunized mice
(33,47)
and CD4 T cells
(13)
.
Using SEM, Boulos et al.
(48)
examined the effect of
vaccination of mice with the soluble portion (S3)
of large particle fraction antigen of T. spiralis, and
they reported loss of integrity of larval cuticle,
some were attacked by granulocytes and few
36 Nassef et al.,
appeared completely destructed with loss of normal
architecture.
Acknowledgement: The authors would like
to thank staff members of Pathology Department,
Faculty of Medicine, Menoufiya University,
Anatomy Department, Faculty of Medicine, Ain
Shams University and Electron Microscopic Unit,
Faculty of Science, Alexandria University for their
kind help and assistance.
Author contribution: NE Nassef designed the
research and revised the manuscript. MM El-Sobky
initiated the research idea, prepared the antigens,
interpreted the results, reviewed the literature, wrote
the manuscript and supervised and assisted AF Afifi
in the practical work.
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Correspondence to
Mona M El-Sobky, MD
Parasitology Department,
Faculty of Medicine,
Menoufiya University
E- mail: [email protected]
38 Nassef et al.,
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Worm and Larval Burden, Histopathological and Ultrastructural Evaluation of T. Spiralis Vaccination Using Crude Worms And/or Larvae Antigens: Experimental Studies

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Trichinellosis is a serious parasitic zoonosis

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