IMMUNODIAGNOSTIC STUDIES ON TOXOCARA VITULORUM IN RABBITS

Dept. of Parasitology,

Fac. Vet. Med., Beni-SuefUniv.

Immunodiagnostic studies on Toxocara vitulorum in rabbits

(With 3 Tables and 4 Plates)

By

W.M. Mousa; Kh.M. El-Dakhly* and H.S. Lotfy**

* Dept. of Parasitology, Fac. Vet. Med., Beni-SuefUniv.

** Animal Health Research Institute, Beni-Suef

(Received at 19/5/2008)

دراسات مناعية تشخيصية علي التوکسوکارا فيتلورم في الأرانب

وحيد محمد علي موسي ، خالد محمد الداخلي ، حسين سيد لطفي

في هذه الدراسة تم تشخيص التوکسوکارا فيتلورم في الأرانب وکذلک محاولة تحصينها، ولهذا تم تفسيم عدد 12 من ذکور الأرانب النيوزيلاندي الي ثلاث مجموعات متساوية، حيث کانت المجموعة الأولي (أ) مجموعة محصنة بمستخرج البويضات المعدية وتم عدوي المجموعة الثانية (ب) ببويضات النوکسوکارا فيتلورم بدون تحصين في حين تم إعطاء المجموعة الأخيرة (ج) الحاوي adjuvant فقط واعتبرت کمجموعة ضابطة سلبية في. وقد أجريت عدة اختبارات مناعية علي المجموعات الثلاث، شملت اختبار الأليزا واختبار اللطخ المناعي واختبار التکسير البروتيني لجزيئات مولد الضد المستخدم عن طريق الفصل الکهربي ، حيث اوضحت نتائج الأليزا للمجموعة الأولي أن ظهور أول مستوي ايجابي للأجسام المناعية المضادة (0.623) بدأ عند اليوم الـ 14 من التحصين ووصل الي أعلي مستوي له (0.936) عند اليوم الـ 21 من العدوي. وفي المجموعة الثانية کان أول ظهور للأجسام المضادة (0.496) عند اليوم الـ 21 من العدوي في حين کان أعلي مستوي لها (0.657) عند اليوم الـ 30 من العدوي. وأخيرا في المجموعة الثالثة بدا ظهور اول مستوي ايجابي (0.418) عند اليوم الـ 21 من التحصين وصولا الي أعلي ترکيز (0.778) عند اليوم الـ 30 من العدوي. وبالنسب لنتائج الفصل الکهربي الجزيئي لبروتينات مولد الضد المستخدم وجدت 8 حزم بروتينية علي مدار التجربة وهي علي الترتيب 240 و 135 و 40 و 33 و 28 و 24 و 22 و 18 کيلو دالتون.  وبالنسبة لأختبار اللطخ المناعي وجدت أيضا 6 حزم بروتينية مختلفة وهي 43 و 40 و 33 و 24 و 22 و 18 کيلو دالتون. هذا وقد وجدنا ان الحزم 33 و 24 و 18 کيلو دالتون قد ظهرت طوال فترة التحصين والعدوي أيضا مما يجعلنا نعتبرها حزم حامية يمکن استخدامها في ايجاد التحصين المناسب. ويجب أن نشير الي أن الحزم البروتينية 43 و 40 و 33 و 24 و 22 و 18 قد ظهرت مبکرا بينما ظهرتالحزم البروتينية 50 و 60 کيلو دالتون متأخرة في نهاية التجربة. وبالنسبة للتغيرات الباثولوجية فقد أکدت ما وجدناه حبث ترکزت في أکباد ورئات الحيوانات المعدية فقط حيث ظهرت البقع البيضاء بکثرة في الکبد وامتلأت الرئة بالبقع والعقد النزفية في حين أنه ظهرت هذه الأعضاء أقرب الي الشکل الطبيعي في المجموعة المحصنة.

summary

In the present study, serodiagnosis as well as trial for vaccination of Toxocara vitulorum were done. Twelve male New Zealand rabbits were divided into 3 equal groups; Toxocara vitulorum immunized group (A), control positive Toxocara vitulorum infected group (B) and control Freund’s adjuvant group (C). Enzyme-linked immunosorbent assay (ELISA), Western Blot and protein fractionation by using Sodium Dodecyl Sulphate Gel Electrophoresis (SDS-PAGE) were done. Concerning ELISA results, Toxocara vitulorum-immunized group showed the first positive mean antibody titre (0.623) at the day 14 post immunization and reached the peak (0.936) at the day 21 p.i. In control infected group, the first positive mean antibody titre (0.496) appeared at the day 21 p.i and reached the peak (0.657) at the day 30 p.i. In control adjuvant group, the first positive mean antibody titre (0.418) appeared at the day 21 post immunization and reached the highest level (0.778) at the day 30 p.i Concerning  results of SDS-PAGE, electrophoretic analysis with silver stain revealed 8 protein bands with molecular weights 240, 135, 40, 33, 28, 24, 22 and 18 kDa, respectively. The analysis of immunoblotting cleared polypeptide bands of molecular weights 43, 40, 33, 24, 22 and 18 kDa in different sequences along the experiment. It is concluded that, protein bands of molecular weights 33, 24 and 18 kDa appeared along the course of immunization and still also post infection. Therefore, they are considered as protective bands that can be used for preparation of a purified vaccine. It is worthy to mention that protein bands of molecular weights 43, 40, 33, 24, 22 and 18 kDa were detected early (early diagnostic bands); while those of molecular weights 60 kDa and 50 kDa were detected at the day 45 p.i (late diagnostic bands). Concerning to the histopathological results, lesions were prominent in liver and lungs of control infected group showing several hepatic milky spots as well as diffuse eosinophilic infiltration in addition to haemorrhagic patches and nodules in lung tissues. The picture in immunized group was greatly reduced appearing more or less normal.

Key words: Toxocara vitulorum, immunodiagnosis, rabbits

Introduction

Toxocara vitulorum is a pathogenic parasite of the small intestine of buffaloes. It is common to find buffalo calves highly infected between 15 and 90 days of age (Starke-Buzetti et al. 1983) and the main way of infection of the calf is via the transmammary route in neonatal calves (Starke-Buzetti et al. 1992). The main route for migration of                  T. vitulorum larvae in the buffalo host is from the gut to the liver by passive blood transport through the portal vein and in a minor way, through mesenteric lymph nodes. In the resistant host, cows for instance, T. vitulorum larvae may reach the muscles where they survive, but do not molt, possibly as hypobiotic larvae (Omar and Barriga, 1991). Migration of larvae of T. vitulorum in white mice, manifested a behavior similar to other ascarids (Chauhan and Pande, 1972).

The adult parasites are relatively easy to remove from the intestines by chemotherapy, while the larvae are difficult to kill mainly when they are in the musculature and in the brain (Abo-Shehada and Herbert, 1984). Therefore, an alternative possibility of control is to immunize the dams to kill their tissue larvae before they are transferred to the calves. An immunological approach to the control of T. vitulorum has always been attractive, and it would be most advantageous if the larvae could be killed in less vital tissues, preferably the intestine (Barriga and Omar, 1992). However, little is known about the immunological responses of water buffalo against T. vitulorum 

Amerasinghe et al. (1984) found anti-T. vitulorum antibodies in the serum of swamp buffalo cows before and after parturition and also in their colostrum after parturition. Their calves showed the same serum antibodies after birth, but they developed patent T. vitulorum infection. Later, Rajapakse et al. (1994 a) demonstrated antibodies against the excretory/secretory antigens of T. vitulorum larvae (E/S) in serum and colostrum of buffalo cows by ELISA (enzyme-linked immunosorbent assay). High titers of anti-larval E/S antibodies in buffalo cow colostrum and in the serum of their suckling calves, correlated with low                 T. vitulorum fecal egg counts in the calves and suggested that antibodies might have a protective role against activated or migrating larvae. Furthermore, Rajapakse et al. (1994 b) confirmed antibody-mediated protection against T. vitulorum larvae by the ability of buffalo serum or colostrum to inhibit migration of T. vitulorum larvae in mice. Besides, Amerasinghe et al. (1992) reported that, immunization of mice with      T. vitulorum-perienteric antigen induced 100 % and 79 %, respectively, of inhibition of larval migration to their tissues, suggesting a potential protection against the infection. 

The present study aims to immunize rabbits against embryonated egg antigen of Toxocara vitulorum together with using the possible immunodiagnostic techniques (SDS-PAGE, ELISA and Western Blot) for detection of time of peak antibody release and specific diagnostic polypeptide bands. 

MaterialS and Methods

1. Collection and embryonation of eggs:    

Adult Toxocara vitulorum worms were obtained from the slaughtered buffalo calves at Beni-Suef abattoirs, transported to Beni-Suef laboratory of Animal Health Research Institute, washed several times with saline and examined under microscope. Fertile eggs were obtained from uteri of the gravid females using blunt scissor, then, sieved, washed and precipitated several times using 1 % formol-saline followed by keeping in refrigerated adequate solution till use (Sabry, 1999).

The collected eggs were divided into several amounts put in clean petri-dishes and incubated at 28 °C, underwent several rewashing and good aeration followed by microscopical examination to observe the developmental changes and left to embryonate for 30 days (Omar and Barriga, 1991 and Mousa et al., 2001). Some of the embryonated eggs were used for antigen production, and others were kept at 4°C for challenge.

2. Preparation of Toxocara vitulorum antigen:

The embryonated infective T. vitulorum eggs were washed several times with 0.01 M PBS (pH 7.4) by centrifugation at 1500 rpm. for 10 minutes to remove remnants of the formol-saline. Eggs were mixed with an equal volume of the solution, then homogenized at 6000 rpm for 5 minutes in ice bath. The homogenized samples were sonicated for 5 minutes at 5 pulse rate and 60-80 amplitude value using cole parner ultra sonic sonicator. Then the suspension was subjected to high speed centrifugation 14,000 rpm at 4 Cº for 30 minutes, then the supernatant containing soluble antigenic materials was separated and stored at -20 Cº in plastic vials till used (Sabry, 1999 and Arafa, 2008). Protein content of the antigenic components was determined according to Lowry et al. (1951).

3. Rearing and housing of rabbits:

            Twelve male New Zealand rabbits of 1-1.5 kg body weight free from parasitic, bacterial or viral diseases were obtained, reared in clean and disinfected battery, then feed on maintenance ration provided with coccidicidal drugs. Temperature and ventilation were adjusted.

4. Experimental design:

4.1. Grouping of animals:

Rabbits were divided into 3 equal groups:

- Group A (Immunized group): Rabbits immunized against T. vitulorum    antigen.

- Group B (Control positive group): Rabbits were only infected with   embryonated eggs of T.vitulorum used without immunization.

- Group C (Control adjuvant group): Rabbits administered Freund’s   adjuvant only.

4.2. Protocol of immunization:

In groups (A and C), animals were administered complete Freund’s adjuvant (600 µg protein) followed by incomplete one (400 µg protein) as the method described by Mousa et al. (2001).

4.3. Procedure of infection:

Each rabbit (of groups A and B) was challenged after the day 30 from the beginning of the experiment with 10000 embryonated              T. vitulorum eggs orally using stomach tube.

4.4. Blood collection:

            Blood was collected from the ear veins at the days zero, 14 and 21 post immunization and at the days 14, 21, 30 and 45 p.i., then serum was obtained by centrifugation and preserved at -20 Cº till use.      

5. Diagnostic tools:

5.1. Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis: (SDS-PAGE):

            It is used for determination of molecular weights of polypeptide bands obtained by fractionation of T. vitulorum embryonated egg antigen according to Laemmeli (1970).

5.2. Enzyme-linked immunosorbent assay (ELISA):

            ELISA was applied to detect antibodies against T. vitulorum antigen using 96-well flat microtiter plate according to Starke-Buzetti   et al. (2001) and Arafa (2008).

5.3. Western Blot technique:

            Polypeptide bands obtained from SDS-PAGE were electrophoretically transferred to nitrocellulose according Towbin et al. (1979) and the results were analyzed using Gel pro-analyzer 3.1.

6. Histopathological techniques:

            Pieces from liver and lungs of the sacrificed rabbits were fixed in 10 % formalin, embedded in paraffin sections of 5 µm thickness, stained with haematoxylin and eosin and examined microscopically according to Bancroft and Stevens (1996).

Results

1. Evaluating the egg antigen of Toxocara vitulorum using ELISA:

ELISA was performed on sera of different rabbit groups with                T. vitulorum egg larval antigen at zero day, at days 14 and 21 post immunization, then at days 14, 21, 30 and 45 post infection (p.i.). It has been found that, in Group (A); the first positive antibody titer (0.623) was found at the day 14 post immunization, increased to (0.925), (0.933), (0.936), (0.926) and (0.872) at the day 21 post immunization, 14, 21, 30 and 45 days p.i. respectively. In group (B); the first positive mean antibody titer (0.496) was at the day 21 p.i., increased to (0.657) at the day 30 and became (0.566) at the day 45 p.i. Moreover, in Group (C), the first positive mean antibody titer (0.418) at 21 days post immunization, followed by (0.480), (0.598), (0.778) and (0.743) at the days 14, 21, 30 and 45 p.i. respectively (Table 1).  

It’s worthy to clarify that, ELISA with T. vitulorum egg antigen showed sensitivity reached to 100% since all infected and immunized rabbits were positively reacted.

2. Characterization of T. vitulorum egg antigen by using SDS-PAGE:

Electrophoretic analysis of infective T. vitulorum egg antigen with SDS – PAGE after staining with silver stain revealed eight bands with molecular weights 240, 135, 40, 33, 28, 24, 22 and 18 kDa (Table 2 and Plate 1).  

3. Detection of T. vitulorum specific epitopes by using Western Blot:

Reaction of T .vitulorum antigen with sera of rabbits in group (A) at day zero of the experiment did not reveal any bands due to absence of the specific antibodies against T. vitulorum, while the same reaction revealed three polypeptide bands of molecular weights 33, 24, and 18 kDa at the day 14 post immunization. The same bands appeared at the day 21 in addition to bands of 43, 40 and 22 kDa. Furthermore, six bands of molecular weights 43, 40, 33, 24, 22 and 18 kDa appeared at the day 15 p.i. (post infection). The same bands appeared at the days 30 and 45 p.i. in addition to bands of molecular weights 60 kDa and 50 kDa. It’s worthy to mention that bands of 33, 24 and 18 kDa appeared along the course of the immunization and still post infection, so it may be the protective bands from which the purified vaccine may be prepared. Bands of molecular weights 43, 40, 33, 24, 22, and 18 kDa appeared early so, they may be considered as early diagnostic bands, while bands of 60 kDa and 50 kDa appeared at the day 45 p.i., so may used as specific diagnostic bands for the late infection (Table 3 and Plate 2).

4. Histopathological findings:

The pathological findings were prominently in liver and lungs due to the pattern of the migratory behaviour. In the immunized group (group A), gross lesions revealed relative lung resistance and more or less normal hepatic configuration, while microscopic findings of these organs showed mild eosinophilic granulomas in the liver associated with normal lung outline.

On the other hand, in Toxocara vitulorum infected group (group B) milky spots were seen on liver surface as well as haemorrhagic patches in the lungs, while the microscopic picture revealed eosinophilic infiltration in both hepatic parenchyma and lung tissues (Plates 3 & 4).

Table 1: Evaluation the T. vitulorum eggs antigen for capture of their                 antibodies in different groups using ELISA.

         (Cut off value = 0.410)

Table 2: Characterization of infective T. vitulorum egg antigen with SDS-PAGE.

* Amersham  prestained  broad range molecular weight protein marker  (Bio-lab Company).

Table 3: Determination of specific epitopes of T. vitulorum antigen with sera of group (A) by using Western Blot.  

                 p.imm (meaning post immunization)

Plate 1: A, (A) Showing the polypeptide bands revealed from the electrophoresis of T. vitulorum  eggs using  SDS –PAGE

Plate 2: B, (B) showing polypeptide bands revealed from the reaction of T. vitulorum with sera of group (A)

(3), (4), (5), (6) and (7) revealed days 14 post. Imm., 21 post. Imm., 14 p.i, 30 p.i and 45 p.i

Plate 3: Macroscopical picture of both immunized and infected groups        A) Viscera of a rabbit in group (A) showing more or less normal     organs

            B) Liver of a rabbit in group (B) showing milky spots

            C) Lung of a rabbit in group (B) showing nodules

Plate 4: Histopthological picture of both immunized and infected groups

A) Liver in group (A) showing mild eosinophilic infiltration (H & E x 400)

B) Lung in group (A) appeared more or less normal (H & E x 400)

C) Liver in group (B) showing severe eosinophilic infiltration (H & E x 400)

D) Lung in group (B) showing diffused eosinophilic granulomatous reactions (H & E x

     200, small square x 1000)

Discussion

Concerning ELISA results to evaluate the infection by using soluble extract of Toxocara vitulorum antigen, it was evident that, in the immunized group, the first positive mean antibody titre (0.623) appeared at the 2^nd week post immunization; while it appeared in the 2^nd and the 3^rd weeks p.i. in both group C (0.480) and group B (0.496) respectively. These results agreed with those obtained by Selim et al. (1986) who revealed that the mean positive antibody titre in mice experimentally infected with Toxocara vitulorum eggs by using the whole worm antigen, appeared at the 2^nd weeks p.i. Similar results were obtained by Ferrnado et al. (1986) who used an embryonated egg antigen for detection of antibody level against Toxocara vitulorum infection in cows by using ELISA. Results of Khalil et al. (1989) showed that the embryonated egg antigen was more sensitive than the adult worm antigen using ELISA where it gave the highest percentage of positivity.

Furthermore, ELISA was highly sensitive against toxocariasis by using embryonated egg antigen. It was found that all infected and immunized rabbits showed positive results (100 % sensitivity) using Toxocara vitulorum antigen. Efficacy of ELISA as a diagnostic tool was supported by Cypess et al. (1977)who reported that ELISA was highly sensitive in diagnosis of toxocariasis. Extraction of embryonated Toxocara vitulorum antigen was found to be more sensitive, since it was used for ELISA giving the highest positive results (Khalil et al. 1989 and Arafa 2008). In addition, Nunes et al. (1999) revealed 100 % sensitivity after 20 days post inoculation in mice, so the used embryonated egg antigen was characterized to be highly sensitive against toxocariasis by using ELISA. Starke-Buzetti et al. (2001)could detect antibodies against natural infection of Toxocara vitulorum in water buffaloes by ELISA using soluble extract antigen of the nematodal larvae. Moreover, Usha et al. (1997)detected that circumlarval precipitin (CLP) test in buffaloes gave maximum antibody titre at day 14 p.i. Abdel-Megeed and Abdel-Rahman (2003)used 5 antigens in diagnosis of Toxocara vitulorum in buffalo calves using ELISA; they reported that egg antigen was the second potent antigen.

Concerning data of characterization of embryonated egg antigen of Toxocara vitulorum by using SDS-PAGE, it was evident that bands of molecular weights 240, 135, 40, 33, 28, 24, 22 and 18 KDa were found respectively. More or less similar results were obtained by Starke-Buzetti and Ferriera (2004) who revealed bands of molecular weights 190, 150, 110, 90, 64, 56, 48 and 19 kDa when they used excretory/secretory (E/S) antigen of Toxocara vitulorum larvae; the slight variation in the revealed bands might be referred to the type of the antigen used. Results obtained by Abdel-Megeed and Abdel-Rahman (2003) coincided with the present findings, where they detected polypeptide bands of molecular weights 137.7, 81, 75, 48 and 21 kDa. On the contrary, Abdel-Rahman (2000) revealed only 2 polypeptide bands of 92 kDa and 87 kDa when antigen of adult worm is analyzed by SDS-PAGE. This variation might be related to the difference of antigen used. He added that ELISA was more potent with using this antigen and gave 100 % sensitivity. Moreover, Ferreira and Starke-Buzetti (2005) used SDS-PAGE on perienteric fluid antigen obtained from buffalo calves and revealed nine bands of 11, 14, 38, 58, 76, 88, 112 and 165 kDa. They added that the last 3 bands remained during the peak of egg output.

Concerning results of western blot, five bands were recorded at the day 15 p.i. and these bands were 43, 40, 33, 24, 22 and 18 KDa and were identified as early diagnostic bands, and those of 60 and 50 KDa appeared at both 30 and 45 days p.i and they were identified as late diagnostic bands. Similar results were revealed by Barriga and Omar (1992)who stated that specific bands of soluble extract of embryonated eggs antigen against sera of rabbits experimentally infected with Toxocara vitulorum by using western blot appeared at day 15 p.i.

Concerning histopathological findings, rabbits of group (B) showed milky spots in the hepatic parenchyma and the pulmonary tissues showed haemorrhagic patches and some nodules. Unlikely, rabbits of group (A) revealed both normal liver architecture and more or less normal lung tissue indicating pulmonary resistance. Similar results were obtained by Gorp et al. (1987)who detected that mice experimentally infected with Toxocara vitulorum showed no or a low degree of cellular reactions but macroscopic hemorrhages might be observed. Also, Hussein et al. (1980) recorded no lesions in rats inoculated with Toxocara vitulorum eggs, and only few larvae were detected in their livers and lungs. Moreover, Abeydeera and Roberts (1991) evaluated the response of liver tissue in buffaloes experimentally infected with Toxocara vitulorum larvae, by which the lesions produced varied from mild cellular infiltrations in early stages to eosinophilic granulomas with degenerating larvae in later stage of the infection. On studying the course of experimental infection of rabbits with Toxocara vitulorum embryonated eggs Omar and Barriga (1991) revealed that size of muscle larvae did not develop beyond the infective stage suggesting that they were hypobiotic larvae. Furthermore, Barriga and Omar (1992)concluded that, 3 times infections of rabbits with Toxocara vitulorum revealed that larvae declined steadily in liver and lungs from day 5 to 30 of the 1^st infection, was absent in liver at the day 30 of the 2^nd infection, and in both organs in day 30 of the 3^rd one. In addition, Pramanik et al. (1994)reported that infection of rabbits with Toxocara vitulorum showed the highest recovery in liver then in lungs; and larval recovery was highest at day 3 p.i and declined to the lowest one at day 63 p.i. 

It is worthy to mention that Srivastava et al. (1988)studied the histopathological findings in mice experimentally infected with Toxocara vitulorum eggs and found that migration of the larvae lead to degeneration of the hepatic cells together with leucocytic infiltration along the migratory tracts. The results confirmed that the migratory pattern of Toxocara vitulorum is closely related to that of Toxocara canis. 

Finally, vaccine efficacy was evaluated by both positive antibody titre and the hepatic eosinophilic granulomas formation. These findings coincided with those obtained by Amerasinghe et al. (1992)showed that mice exposed to three or more infections with Toxocara vitulorum eggs lead to protection against the challenge. Following parentral immunization of mice with variety of Toxocara vitulorum soluble antigens (extract, E/S or perienteric) from adults, larvae induced statistically significant protection against the infection. They added that, the more immunogenic component was adult perienteric fluid (100 % protection) followed by larval E/S (92 % protection). They proved that oral infection of mice on 3 or more occasions with Toxocara vitulorum eggs induced protection against a challenge infection of the parasite. Besides, Rajapaske et al. (1994 a) demonstrated antibodies against E/S antigen of Toxocara vitulorum in serum and colostrum of buffalo calves by using ELISA and confirmed antibody-mediated protection against Toxocara vitulorum larvae by the ability of buffalo serum or colostrum to inhibit migration of Toxocara vitulorum larvae in mice.

Furthermore, Rajapaske et al. (1994 b) suggested that antibodies detected in mice immunized against Toxocara vitulorum might adversely affect the metabolism of larvae through antibody-mediated cell mediated cytotoxicity, where antibodies might bind to the surface of the migrating larvae.

Further studies must be done to find out more specific epitopes derived from Toxocara vitulorum embryonated eggs and/or larvae in order to obtain more specific diagnostic bands that facilitate the arrival to appropriate diagnosis from which the proper vaccine can be obtained.   

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