Dept. of Parasitology and Animal Diseases

National Research Centre, Giza, Dokki, Egypt.

Structural characterization of Fasciola gigantica partially purified worm antigen and its potency in diagnosis

 of fascioliasis

(With One Table and 5 Figures)

By

Kadria N. Abdel Megeed

(Received at 29/3/2005)

الوصف الترکيبى لمولد الضد المنقى جزئياً من ديدان الفاشيولا جيجنتيکا

وقدرته على تشخيص مرض الفاشيولا

قدرية نصر عبد المجيد

اشتمل هذا البحث على عمل تنقية جزئية عن طريق جهاز التحليل الکروماتوجرافى للمستخلص الخام للطور البالغ لديدان الفاشيولا جيجينتيکا باستخدام سيفدکس G200 وقد نتج عن هذا التحليل فصل 4 أجزاء (مرکبات) من هذا المستخلص الخام. وقد أظهر جهاز الفصل الکهربائى (electrophoresis) صورة بسيطة لکل من هذه الأجزاء الأربعة مقارنة بصورة المستخلص الخام. وقد تم اختبار الکفاءة المناعية للأجزاء الأربعة المفصولة من المستخلص الخام مع الأجسام المناعية المحضرة فى الأرانب باستخدام اختبار الاليزا وقد أظهر هذا الاختبار تفوق المرکب الثانى (F2). وقد نجح هذا المرکب (F2) فى تشخيص مرض الفاشيولا بنسبة 100%. وقد تم عمل وصف ترکيبى لهذا الجزء (F2) عن طريق استخدام جهاز الفصل الکهربى (electrophoresis) ونقطة التعادل الکهربى (isoelectric point ) وأيضاً التحليل الأمينى لهذا المرکب وقد أظهر التحليل الکهربائى أن هذا الجزء (F2) يحتوى على ثلاثة مرکبات ذات أوزان جزيئية 100، 106، 133 کيلو دالتون وکانت نقط التعادل الکهربى لهذه المرکبات هى 5.2 ، 5.9 ، 6.5 ، 7.2 وقد أظهر التحليل الأمينى لهذا المرکب (F2) أنه يحتوى على 17 حمض أمينى وکانت نسبة Lysine و Alanine و Proline کبيرة مقارنة بباقى الأحماض الأمينية.

SUMMARY

Chromatographic analysis of Fasciola gigantica adult crude extract was undertaken using Sephadex G-200. Four fractions were isolated by this approach. The isolated fractions showed simple electrophoretic profile, as judged by SDS-PAGE, compared to the complex profile of crude extract. Fraction 2(F2), exhibited the most potent activities against rabbits hyperimmune serum in ELISA. This fraction was utilized in diagnosis of fascioliasis among buffaloes recording 100% sensitivity. Structural characterization of the isolated fraction, F2, by SDS polyacrylamide gel electrophoresis, isoelectric focusing and amino acid analysis showed that F2 consists of only three polypeptides of 133 KD, 106 KD and 100 KD with isoelectric points of 7.2, 6.5, 5.9 and 5.2. Moreover, 17 amino acids were identified in F2 with high proportions of only three of them (Lysine, Alanine and Proline).

Key words: Fasciola gigantica, gel filteration, ELISA, diagnosis.

Introduction

            Fasciola species are parasitic trematodes with a world wide distribution, which infect a wide variety of mammals, including humans (Mezo et al., 2003). Infection with Fasciola gigantica is considered one of the most causes of economic loss of animals in Egypt.

            The parasitological diagnosis of fascioliasis based on the demonstration of the eggs in feces is usually unsatisfactory due to false passage of eggs and failure of immature worm to maturation. Disease occur as early as 3 weeks post infection while parasitological diagnosis is only possible at about 10-14 weeks after infection when eggs begin to appear in feces (Hillyer, 1981; Ibarra et al., 1998 and Hasseeb et al., 2003). So, serological diagnosis is reliable and potent alternative approach to overcome the deficiencies of fascioliasis diagnosis by parasitological means (Hillyer, 1981 AND Hillyer et al., 1992). Also the advantage of the serological tests is its capability to detect IgG anti Fasciola antibodies from the second week post infection , which is remarkably early in the diagnosis of this diseases (Ibarra et al.,1998).Trials have been proposed  serodiagnosis of F.gigantica  infection in ruminants (Swarup et al, 1987 and Fagbemi  & Obarisiagbon, 1990). Serodiagnosis of fascioliasis is carried out by ELISA (Clery et al., 1996; Sanchez-Andrade et al., 2000; Paz-Silva et al., 2003 and Velusamy et al., 2004).

            The current study aims to partially purified antigen from Fasciola gigantica by gel filteration chromatography. The resulting fractions analyzed by ELISA in order to evaluate the most sensitive fraction and assess its potency in diagnosis of fascioliasis among naturally infected buffaloes. Also, identifying the structural characterization for the most potent fraction.

Materials and Methods

Parasites: Fasciola gigantica adult worms were collected from condemned livers of buffaloes slaughtered in Cairo abattoir.

Antigen preparation: F. gigantica adult worms were washed thoroughly with distilled water to eliminate traces of bile and blood. The antigen was prepared by minor modifications of the method described by Nordon and Strand (1984). One volume adult worms were suspended in three volumes in lysis buffer. Suspension of the parasite was homogenized in an ice bath. Homogenate was sonicated at 28m at 60 pulses in an MSE sonicator. The sonicate was centrifuged at 10.000 g for 1 hr and supernatants collected, assayed for protein content by method of Lowry et al. (1951) and aliquoted and stored at –20°C until use.

Rabbit hyperimmune serum: The rabbit immune serum was prepared according to the technique adopted by Fagbemi et al. (1995). Briefly, rabbits were immunized subcutaneously with @ 40 mg of whole worm extract of F. gigantica in Freund’s complete adjuvant. A booster dose of @ 40 mg in Freund’s incomplete adjuvant was given on day 14. Second and third booster doses were given on day 21 and 28 and serum samples were collected 4 days after last immunization.

Buffalo sera collection: Sera from 54 naturally infected buffaloes were collected and analyzed for antibodies detection. The infection was ascertained by coprological examinations according to Thienpont et al. (1979). 37 sample sera from coprologically negative buffaloes were also collected and analyzed for antibody detection. Buffaloes sera were heat inactivated at 56°C for 30 minutes for decomplementation before analysis.

Gel filteration chromatography: Sephadex G-200 column was used for the fractionation of F. gigantica extract. The column was equilibrated and eluted with 0.05 M PBS containing 0.02% NaN₃. The flow rate was 12 ml/h and fractions were collected in 1 ml aliquots. Column eluates were monitored at 280 nm. The column was calibrated with molecular weight standards from Bio-Rad Laboratories. Blue dextran (2000 KD) was used to determine the void volume.

Enzyme linked immunosorbent assay (ELISA):

ELISA was adopted to evaluate F. gigantica fractions activities utilizing rabbit hyperimmune sera according to Santigo et al. (1986). It was also used to evaluate the potency of the selected fraction in fascioliasis diagnosis. The assay was performed as described by Wijffels et al. (1994). The optimum antigen concentration and sera dilution were determined by checkerboard titration. ELISA plates were coated with 50 mg/ml of each antigen. The selected dilution of sera was 1:100. Antibovine IgG horse-radish peroxidase conjugate was used. ELISA OD cutoff values were calculated by the method of Allan et al. (1992).

SDS-polyacrylamid gel electrophoresis (SDS-PAGE):

20 mg of crude extract and also of pure fractions collected after gel filteration were electrophoresed, under reducing conditions, on 7.5% SDS-PAGE as described by Laemmli (1970). Gel was stained with silver stain according Wray et al. (1981). High and low molecular weight standards were electrophoresed in the same gel.

Isoelectric focusing (IEF): IEF of the isolated fraction was performed as described by O’Farrell (1975) in slab gel supplemented with urea and ampholine. Gels were stained with commasie blue and photographed wet. Isoelectric focusing of a particular protein can be determined by mixing a mixture of proteins of known isoelectric points on the same gels ranged from 4.9-8.4.

Amino acid analysis: Free amino acid composition of the isolated fraction was determined after hydrolysis in HCl prior to analysis with amino acid analyzer (GBC Australia).

Results

Fractionation of F. gigantica crude extract on sephadex G-200 column chromatography:

            Gel filteration was adopted for the fractionation of F. gigantica extract. The elution profile is shown in Fig. (1) where four fractions were eluted from the column at different absorbance values and elution volumes. Each fraction was collected lyophilized and analyzed separately.

Electrophoretic profile of F. gigantica crude and pure antigens:

            The four isolated fractions were separately electrophoresed on SDS-PAGE. At the same time, F. gigantica crude extract was electrophoresed under the same conditions for comparative purposes. The crude extract was resolved into 12 component (Fig. 2 Lane. A). While the four separated fractions showed simple electrophoretic profile, Fig. 2 Lanes B, C, D. and E.

Antigenic activities of isolated fractions:

            The antigenic activities of each fraction was evaluated by ELISA in which rabbit hyperimmune serum was utilized. As shown in Fig. 3, fraction number 2 showed the most potent activities compared with the three other fractions. Based on these results, fraction number 2 was selected to diagnose fascioliasis among naturally infected buffaloes.

Parasitological examination:

            A total of 91 buffaloes slaughtered in Cairo abattoir were examined parasitologically for detection fascioliasis. The examination revealed 54 buffaloes infected with Fasciola gigantica and 37 buffaloes gave negative results. 

Diagnosis of buffaloes fascioliasis by F. gigantica fractions 2:

            Sample sera collected from naturally infected and noninfected buffaloes, as proved by coprological examination, were assayed against fraction 2 in ELISA. The assay confirmed the infection and recorded 100% sensitivity (Fig. 4). The cutoff value was 0.23.

Structural characterization of fraction 2:

a) SDS- polyacrylamide gel electrophoresis (SDS-PAGE): The electrophoretic profile of the isolated fraction 2 is shown in (Fig. 2. Lane C). This fraction was resolved, under reducing conditions, into 3 bands of molecular weight of 133KD, 106KD, 100KD.

b) Isoelectric focusing (IEF): For further characterization of the partially purified fraction, isoelectric point of the fraction was identified by isoelectric focusing technique. As shown in Fig. 5, the components have PIs of 7.2, 6.5, 5.9 and 5.2.

c) Amino acid analysis:  For additional structural characterization of the isolated fraction 2, analysis of its free amino acids was undertaken. The fraction exhibited 17 amino acids as shown in table 1. The fraction is rich in Lysine (8), Alanine (7.5) and Proline (5.2) While Isoleucine (0.6), Methionine (0.8) were present in minute amounts.

Discussion

            Immunodiagnosis of parasitic infection faces the problem of cross-reaction. Production of purified antigen (s) is only way to minimize the cross reactivity.

            In the current research, a partial purification of F. gigantica crude extract by gel filteration using sephadex G200 was undertaken. The purification process resulted in four fractions of different protein content and antigenic activities and the fraction 2 was the most potent fractions as judged by ELISA. Previously, comparable results were obtained, where two fractions were obtained by Carlos et al. (1988). They were purified F. hepatica antigen by gel filteration and HPLC and evaluated the putative potency of both fractions. Rhee et al. (1986) used sephadex G100 column chromatography to obtain the most specific and sensitive fractions from crude antigen of F. hepatica for immunodiagnosis of bovine fascioliasis. Seven fractionated antigens were obtained and the fifth antigen was suggested to be the specific antigen for the immunodiagnosis of bovine fascioliasis. Cervi et al. (1992) revealed that F. hepatica total antigen gave four fractions in a sephadex G-100 column and these fractions showed the presence of different antigenic components. Osman et al. (1992) indicated that four peaks were obtained when Fasciola somatic antigen was fractionated by column chromatography and these fractions evaluated by ELISA.

            The electrophoretic profile of F. gigantica crude extracts as studied by SDS-PAGE, in the current research, exhibited a complex profile of 12 components in both high and low molecular weight ranges (24-205 KD), while the four isolated fractions were showed simple profiles. Electrophoretic make up of Fasciola crude extract by SDS-PAGE was previously probed. Yadav and Gupta (1995) indicated that mature F. hepatica antigen separated out in 9 bands in the range 12-95 KD. SDS analysis of F. hepatica total antigen showed glycoproteins bands ranging from 14 to 94 KD (Cervi et al., 1992). Electrophoretic profile of F. gigantica surface and tegument antigens was showed by Krailas et al. (2002) and they demonstrated that the proteins had molecular weights of 20-97 KD. The contradiction in the results may be account for the difference in the species of parasite or the type of antigen used.

            The majority of indirect diagnostic methods based on antibodies detection are well described in the literatures (Welch et al., 1987). ELISA is considered the most diagnostic method because it is highly sensitive and specific when compared to diagnosing Fasciola species by coprological means, (Ibarra et al., 1998; Fagbemi et al., 1997 and Hasseeb et al., 2003). ELISA have been found to be suitable for diagnosis of fascioliasis due to high sensitivity, possibility of processing many sera samples and its capability to detect IgG anti Fasciola antibodies early post infection (Arriaga de Morilla et al., 1989 and Ibarra et al., 1998). In the present study, as judged by ELISA, fraction 2 (100-133 KD) showed high sensitivity in diagnosing fascioliasis. Where detection of antibody sera collected from 54 buffaloes, proved parasitologically infected with F.gigantica, reactive to the fraction 2 and revealed 100% sensitivity.  Rhee et al. (1986) revealed that fraction number fifth, resulting from F. hepatica fractionation by gel filteration chromatography, had putative potency in the immunodiagnosis of fascioliasis. Carlos et al. (1988) found that a fraction with high molecular weight, 150-160 KD to be very reactive with sera from early fascioliasis.

            The identification of antigens is of fundamental importance, not only to obtain a better understanding to mechanisms of immunity but aslo to facilitate the preparation of purified specific antigen suitable for immunodiagnosis (Carlos et al., 1988).

            The important facet of the present study is the structural characterization of the fraction 2, which was found to be consisted of only three polypeptides of molecular weights 100, 106, 133 KD. Carlos et al. (1988) characterized the separated fractions electrophoretically and found to be of molecular weight ranged from 150-160KD. Espino et al. (1993) reported that the immunogenic purified fraction of F. hepatica had molecular weight ranged from 13KD-37 KD.

            The purified fraction isolated in the present study was also characterized according to its isoelectric points by isoelectric focusing technique. The assay recorded four components of approximately PIs 7.2, 6.5, 5.9 and 5.2. These four components had three molecular weight as showed by electrophoresis analysis introduced the possibility that one of the three polypeptides splitted to two isoelectric points during isoelectric focusing assay. This assumption must be investigated in further studies.

            Identification of the amino acid composition of the pure antigen and its structural details is considered an essential step during purification process (Abdel Rahman, 2000).      Previously, studies related to purification of F. gigantica adult extract have been concerned with evaluation of pure fractions in serodiagnosis. Nevertheless, no definitive information regarding the molecular nature or isoelectric points were introduced.

            So, the current research dealt with further information on the structure of the pure antigen, its free amino acid composition was analyzed. It was concluded that, this fraction is consisted of 17 amino acids with high proportions of only three of them (Lysine, Alanine and Proline). Moreover, there is apparently little amount of Isoleucine and Methionine. Amino acid analysis of Schistosoma mansoni cercarial preparations was previously studied by Caulfield et al. (1987). They observed that threonine, serine and glutamic acids comprised 44% of the amino acid residues of the protein. After purification of cercarial preparations on sepharose column chromatography, free amino acids, predominantly glycine and serine were found to comprise 17% of the total protein. These amino acid presented in low proportion in the current research although both are Trematoda, this may account for the difference in the stage of parasites used in both studies. Cox et al., 1990 observed high amount of glutamic and asparagin in Haemonchus contortus third stage larvae. From these previous studies together with the present one we can detect that, each helminth has its own amino acid structure and unique proportion of each amino acid.

            Further purification of adult worm antigens of F. gigantica would be of significance in the diagnosis process. Also, ELISA technique used in this study offered a diagnostic alternative method for detecting early infection of F. gigantica in animals and it is recommended for seroepidemiological survey for F. gigantica infection.

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Table 1: Analysis of free amino acids of F. gigantica isolated fraction (F2)

Fig. (3) Evaluation of antigenic activities of F. gigantica fractions, resulted from gel filteration chromatography, by ELISA.

Fig. 4: ELISA evaluation of the potency of F. gigantica isolated fraction 2 in the diagnosis of fascioliasis in buffaloes

            Horizontal line show cutoff value

                 Horizontal line show cutoff value

Discussion

Table 1: Analysis of free amino acids of F. gigantica isolated fraction (F2)