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Introduction

Trichinellosis is a serious parasitic zoonosiswith world wide distribution world (1). The causativenematode, Trichinella infects a wide variety ofvertebrates (2). There are eleven Trichinella species, 8of them are encapsulated in host muscle tissues andinfect only mammals as T. spiralis , T. native and T.britovi , while the other 3 species are non encapsulatedand 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 humaninfections (4).

T. spiralis is considered as both an intestinaland tissue parasite. There are two main phasesof infection; enteral (affecting the intestine)and parenteral (outside the intestine) (5). In man,the intestinal phase presents as gastroenteritissimulating food poisoning as early as 24 hoursafter ingestion of infected pork. Larval migrationand penetration of muscles are manifested by fever,eyelids edema, myositis and weakness of involved

Worm and Larval Burden, Histopathological and Ultrastructural Evaluationof T. spiralis Vaccination Using Crude Worms and/or Larvae Antigens:Experimental Studies

Nashaat E. Nassef, Mona M. El-Sobky and Amira F. AfifiParasitology Department, Faculty of Medicine, Menoufiya University, Egypt

27

Abstract Background: Currently, there is no vaccine for T. spiralis ; however, several studies have been made towardsunderstanding 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 wereimmunized 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 adjuvantand phosphate buffer saline before infection. At the 8th day post-infection (PI), 12 mice from each groupwere sacriced and the intestinal worm burden was assessed, while the muscle larval burden was evaluatedat 28th day PI in the remaining mice of each group. Intestinal and skeletal muscle specimens were preparedfor 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 gavesignicant reduction in both intestinal and muscle burdens. Histopathological examination revealed markeddecrease in intestinal inammatory inltrates, and marked reduction of encysted larvae with mild inltrationaround the degenerated larvae in mice of group C. SEM and TEM results conrmed the signicant effectof the combined vaccine (Group C).Conclusion: Vaccination with combined worms and larval antigens gave the most protective action againstT. spiralis challenge infection.

Recommendations: The use of combined antigen in mass vaccination of reservoir animals may decreasethe 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

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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 promisingresults (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 numberof 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

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29Vaccination 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). Ingroup A, each mouse was immunized by intra-peritonealinjection of 200 g (100 g of larval antigen mixed withequal volume of complete Freunds adjuvant (CFA)on the 1st day, followed by two booster doses of 50g of antigen mixed with equal volume of incompleteFreunds adjuvant (IFA) on days 15 and 28 (22). One weekafter the last dose of injection, each mouse was infectedorally 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, dehydratedin 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 ElectronMicroscopic 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 thedifferent 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 showedsignificant reduction in the mean number of adultworms (86%, 73% and 92, respectively) in allvaccinated groups compared with non immunizedinfected control groups (P0.05) (Table1). Using student t test, there was significant reductionof worms in group C compared with groups A andB (P

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30 Nassef et al. ,

marked decrease in inflammatory infiltrates in groupC, while mild to moderate intestinal inflammationswere observed in groups A and B. Compared to group

D (Figure 5), muscle sections showed reduction ofencysted larvae with mild surrounding infiltration,and hyaline degeneration of the larvae. In addition,there was mild muscle degeneration adjacent to thecyst with normal distant muscle. These findingswere obvious in muscles recovered from mice ofgroup C (Figure 6) and varied from mild to moderatein groups A and B (Figures 7 and 8).

Examination of worms and larvae by SEMrevealed that with combined antigen (Figures 11and 12), there was marked destruction of larvae andworms with marked swellings, disintegration of the

cuticles, multiple large blebs and vesicles with lossof normal morphology and architecture of the cuticlecompared to infected control group (D) (Figures 9and 10). Larval and worms antigens gave the sameresults but with lower degree of destruction (Figures13-16). TEM of muscle sections showed mild tomoderate enlargement and increase in number ofmitochondria with moderate depletion of glycogen,slight disturbance of Z line, intact myofillamentswith normal nucleus and mild to moderate dilatationof endoplasmic reticulum. These findings were moreobvious with combined antigen (Figures 19 and 20)

than the other antigens (Figures 21-24) compared tonormal and infected control groups D and E (Figures17 and 18).

Table (1): Global statistical analysis using ANOVA in the studied groups

Table (2): Individual statistical analysis using t-test in the vaccinated groups

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Figure (4): Intestinal section of mousevaccinated with crude adultantigen showing moderate tosevere inflammatory infiltrates inthe submucosa and in the core ofthe villi with ulceration (U) andnecrosis (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 mousevaccinated with combined antigenshowing very small one larvalcyst (C) with degenerated capsule,eosinophilic material replaced thelarvae, mild pre-larval infiltratesand no degenerative changes in msfibers.

Figure (8): Skeletal muscle section of mousevaccinated with adult antigen showingmultiple larval deposition with moderatecellular infiltration around thick capsule

(TC) and moderate to severe degenerationof ms fibers with moderate separation ofms fibers.

Figure (7): Skeletal muscle section of mousevaccinated with larval antigen showingmultiple larval cyst with thin capsule,mild to moderate pre-cyst infiltration

(I), eosinophilic material replaced thelarvae (E) and minimal degeneration ofmuscle fibers.

Figure (1): H & E transverse intestinalsection of infected control mouseshowing severe inflammatoryinfiltrates in the submucosa andthe core of atrophied villi with cutsection of parasite (P) between thevilli and in intestinal lumen.

Figure (2): Intestinal section of mousevaccinated with combined antigenshowing mild inflammatoryinfiltrates with apparently intactmucosa and villi.

Figure (3): Intestinal section of mousevaccinated with crude larvalantigen showing moderateinflammatory infiltrates in thesubmucosa and the core of villiwith less atrophy of villi.

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32 Nassef et al. ,

Figure (16): SEM of T. spiralis larva of mousevaccinated with adult antigen showing

moderate oedema that cause obliterationof the longitudinal furrow, moderate lossof folds, ridges and multiple small blebs.

Figure (15): SEM of T. spiralis adult of mousevaccinated with adult antigen showing

multiple blebs (B), irregular configurationof the cuticle and loss of ridges and folds.

Figure (9): SEM of T. spiralis adult of aninfected control mouse showingcephalic structure of anterior-lateral

papillae (P) found on broad bulkshowing primary folds with largespacing. Fine longitudinal ridges (L),transverse creases (C) and hypodermalglands opening (G) are seen.

Figure (10): SEM of T. spiralis larva ofan infected control mouse showingoral slit (O.S.) like opening whichslightly depressed below the surface,

transverse folds with narrow spacingin between and longitudinal furrowwith no opening or pores on thelateral 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 larvaof mouse vaccinated with larvalantigen showing large swelling(S), moderate oedema, multiple

blebs (B) with area of fissuring (F)and some cuticlular sloughing.

Figure (12): SEM of T. spiralis larva of mouse

vaccinated with combined antigen

showing severe edematous cuticle

with formation of large vesicles (V)

and multiple blebs with loss of normal

features of the cuticle.

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Figure (24): TEM of skeletal muscleof mouse vaccinated with adultantigen showing degeneration ofmyofillament (D.Mf) with enlargedmitochondria (M) that increased in

number, disturbance of Z. line andmarked depletion of glycogen (G).

Figure (22): TEM of skeletal muscleof mouse vaccinated with larvalantigen showing thick collagencapsule (C) of encysted larvainfiltrated with polymorphonuclearleukocytes (P) and the muscle (Ms)adjacent showing no degeneration.

Figure (23): TEM of skeletal muscle of mousevaccinated with adult antigen showingnurse cell of the parasite including

part of the capsule (C) infiltrated withlarge number of plymorphonuclear

cells (P.C) mostly fibroblast andmacrophage surrounding a part of thelarva (L). Part of muscle cells (Ms)showing normal appearance.

Figure (19): TEM of skeletal muscle ofmouse vaccinated with combinedantigen showing slight dilatationof mitochondria (M) with averagenumber, average glycogen (G),normal Z. line, good striation ofmyofillaments, mild to moderatevacculation (V) and average sizeand number of S.E.R.

Figure (20): TEM of skeletal muscle of

mouse vaccinated with combined

antigen showing normal nucleus

of muscle cell, average glycogen

(G), average number and size of

mitochondria (M), interrupted Z.

line and slight vacculation (V).

Figure (21): TEM of skeletal muscle ofmouse vaccinated with larval antigenshowing marked dilatation of S.E.R.,average glycogen (G), disturbanceof Z. line, mild dilatation and ruptureof some mitochondria (M) andmoderate vacculation.

Figure (17): TEM of skeletal muscle ofnormal control mouse showing normalmuscle fibers, normal glycogendistribution (G), good appearanceof Z. line, average size, number anddistribution of mitochondria (M)and normal appearance of smoothendoplasmic reticulum (SER).

Figure (18): TEM of skeletal muscle of infectedcontrol mouse showing encysted larva(L) with highly infiltrated capsule(C) by polymorphonuclear cells (P).Muscle surrounding showing highlydegeneration, loss of striation and Z.

line, loss of glycogen and abnormalshape of muscle cell nucleus (N) withirregular distribution of chromatinmaterials.

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34 Nassef et al. ,

Discussion

Major advances have been made in the pastseveral years towards understanding the immunemechanisms that contribute to host protectionagainst intestinal nematode parasites. Theseinsights may prove useful in the development ofnew immunologically based treatments includingvaccines to enhance resistance to intestinal nematode

parasites (29). Several researches have been done tomodify antigens derived from T. spiralis in order toincrease their specificity and subsequently increasetheir 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 burdenof all vaccinated groups was evaluated. Resultsshowed significant reduction of 86%, 73% and 92%in worm burden in groups A (larval), B (adult) andC (combined), respectively compared to group D(P

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compared with the infected control group D. Otherreports (31,32) confirmed that immunization of miceusing autoclaved T. spiralis larval vaccine with

BCG (adjuvant) and crude larval antigen causedreduction of 94.4% and 89.5%, respectively inmuscle larval burden. On the other hand, lower

percentage reduction in muscle larval burden wasreported in several studies using larval antigen(88% (14), 82.9% (22), 82% (33) and 79% (39) , comparedto 91% in the present study). It was reportedthat oral vaccination with the copolymer micro-encapsulated crude larval extracts and E/S productsinduced stimulation of IF- secretion and inhibitionof IL-4 secretion in spleen and mesenteric lymphnodes of immunized mice (13) . This method inducedconcurrent Th1/ Th2 local and systemic responsesthat are protective, and at the same time may help

balancing the strong Th2 response triggered byhelminth infections. Immunization of mice withDNA vaccine induced both humoral and cellularimmune responses which provided partial protectionagainst 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) usingcrude adult antigen, while another study reported thatmice immunized with worm antigen produced moreresistance to the subsequent challenge infectionleading to significant reduction in worm and musclelarval burden (15). Significantly the combined antigen

produced higher reduction in muscle larvae (96%)in group C than the other antigens in groups A andB (P

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36 Nassef et al. ,

appeared completely destructed with loss of normalarchitecture.

Acknowledgement: The authors would liketo thank staff members of Pathology Department,Faculty of Medicine, Menoufiya University,Anatomy Department, Faculty of Medicine, AinShams University and Electron Microscopic Unit,Faculty of Science, Alexandria University for theirkind help and assistance.

Author contribution: NE Nassef designed theresearch and revised the manuscript. MM El-Sobkyinitiated the research idea, prepared the antigens,interpreted the results, reviewed the literature, wrote

the manuscript and supervised and assisted AF Afifiin the practical work.

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Correspondence toMona M El-Sobky, MD

Parasitology Department,Faculty of Medicine,Menoufiya UniversityE- mail: [email protected]

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