ABSTRACT
Fasciola hepatica and F. gigantica are polymorphic liver flukes that show considerable overlap between species, and various protein separation techniques have been used as alternative means of differentiation. Acid and alkaline polyacrylamide gel electrophoresis (PAGE) show differences between F. hepatica and F. gigantica. Following SDS-PAGE, F. hepatica proteins are characterised by the presence of eight major peptide bands, with molecular weights estimated as 48, 45, 43.5, 37, 33, 29, 27 and 25.5 kDa. In contrast, F. gigantica shows only five major protein bands of 57.6, 54, 48, 29 and 27 kDa. Isoelectric focusing (IEF) demonstrates 17 bands from F. hepatica and 22 bands from F. gigantica between pH 3.5 and pH 10. Although many bands appear common to both species, some are species-specific. Six cases of human acute fascioliasis diagnosed clinically, haematologically and immunologically are also studied. Gel immunodiffusion and immunoelectrophoresis, using adult F. hepatica and F. gigantica antigens, are used to determine the species, and indicate that the antisera are more specific for F. hepatica.
KEY WORDS: Electrophoresis, agar gel. Electrophoresis, polyacrylamide gel. Fasciola hepatica. Fasciola gigantica. Hemagglutination tests. Immunodiffusion. Immunoelectrophoresis.
Introduction
Fascioliasis is a disease that infects a wide range of mammals including man. It is recognised as an important condition by the World Health Organization and recent reports estimate that 2.4 million people are infected. The disease is a serious public health problem in many areas of the tropics and subtropics.
Fascioliasis is caused by hermaphrodite trematodes of the genus Fasciola, with Fasciola hepatica and F. gigantica being the most common.1-4 The fluke is a polytypic species which varies morphologically depending upon the particular host.5,6 Also, a range of morphological types of fasciola occurs in south-east Asian countries such as Japan, Taiwan and the Philippines.7-10
F. hepatica and F. gigantica are easily diagnosed at the extremes of the morphological range; however, intermediate forms also occur and this has led workers to find alternative ways to differentiate them.
Molecular and immunological techniques have been used to differentiate species11,12 and strains of the parasite,13,14 and recently a soluble protein isoelectric focusing technique has been documented, providing simple and reproducible resolution.14
This study aims to differentiate F. hepatica and F. gigantica using native polyacrylamide gel electrophoresis (alkaline- and acid-PAGE), sodium dodecyl sulphate (SDS)-PAGE and isoelectric focusing (IEF) to characterise the body proteins of adult worms in order to identify those that are not typical of either species. In addition, species identification in human cases of fascioliasis will be performed by immunodiffusion and immunoelectrophoresis, using crude extracts of F. hepatica and F. gigantica.
Material and methods
Choice of worms
Worms were obtained from the livers of slaughtered cattle at Alexandria abattoir, Egypt. They were classified as either F. hepatica or F. gigantica by macroscopic appearance and microscopic study of the branching of intestinal ceca. A total of 25 F. hepatica and 55 F. gigantica were studied.
Preparation of crude extract
F. hepatica and F. gigantica were washed in sterile saline and homogenised (10 worms/50 mL saline). The homogenate was centrifuged at 14,000 rpm at 4�C for 1 h. Supernatant protein concentration was measured by the Lowry method15 and found to be 23.5 mg/mL for F. gigantica and 26.5 mg/mL for F. hepatica.
Gel electrophoresis
Native-polyacrylamide gel electrophoresis: Native-PAGE (at pH 8.6 and pH 4.0) was carried out using vertical electrophoresis apparatus (PROTEAN-II Cell, Bio-Rad, Richmond, CA, USA) at 120 V for 2 h. Gels used were 4.5% for stacking and 10% for separation.
For alkaline-PAGE (pH 8.6), TEMED and ammonium persulphate (final concentrations 0.04% [v/v] and 0.07% [w/v], respectively) were added to the stacking gel containing 3.6 mol/L urea and 7.5% (w/v) glycerol in 0.5 mol/L Tris-HCl buffer (pH 6.8). The running gel contained 6.1 mol/L urea with TEMED and ammonium persulphate (final concentrations 0.03% [v/v] and 0.07% [w/v], respectively) in 1.5 mol/L Tris-HCl buffer (pH 8.8). The electrode and running buffer consisted of 0.19 mol/L glycine and 0.24 mol/L Tris (pH 8.6).
For acid-PAGE (pH 4.0), the stacking gel contained 3.6 mol/L urea and 7.3% (w/v) glycerol in 0.5 mol/L KOH adjusted to pH 5.9 with acetic acid; TEMED and ammonium persulphate (final concentrations 0.05% [v/v] and 0.07% [w/v], respectively) were added. The separation gel contained 7.9 mol/L urea, TEMED and ammonium persulphate (final concentrations 0.04% [v/v] and 0.9% [w/v], respectively) in 0.5 mol/L KOH adjusted to pH 2.9 with acetic acid. The running buffer consisted of 0.19 mol/L glycine adjusted to pH 4.0 with acetic acid.16
Sodium dodecyl sulphate polyacrylamide gel electrophoresis: SDS-PAGE was conducted in polyacrylamide gel containing 0.1% SDS according to the method previously described17 and involved denaturation of proteins by heating for 5 min in 1% SDS in a boiling water bath prior to applying them to the gel.
Gel staining
After electrophoresis (PAGE or SDS-PAGE), gels were stained for 2 h with 0.1% (w/v) Coomassie blue R-250 in methanol/acetic acid/water (50:7:43 by volume). Gels were destained with a solution of methanol/acetic acid/water (40:10:50 by volume).
Protein molecular weight determination
Molecular weights (kDa) of fractionated proteins on gels were estimated according to the method of Weber and Osborn18 using standard protein markers (Bio-Rad).
Isoelectric focusing
Six worms of each species were obtained from the livers of infected cattle, incubated at 37� C in PBS for 4 h and frozen at -20�C. When removed from the freezer they were thawed at room temperature, blotted with filter paper and weighed. Worms were then homogenised individually in double-distilled water (3 �L/mg worm) and the homogenate was centrifuged at 11,000 rpm for 12 min.19 Supernatant protein concentration was estimated by the Lowry method.15
IEF was performed on a microprocessor-controlled electrophoresis unit (Phastsystem, Pharmacia Biotechnology, Uppsala, Sweden) as described by Olsson et al.,20 using gels at pH 3.5-10, run at 1500 V, 5 mA and 3.5 W at 15�C. Gel (1-2 mm thick) was prepared using 30 g acrylamide, 0.8 g bis-acrylamide in 100 mL H2O (w/v), 1% TEMED, 0.1% (w/v) riboflavine and 40% Ampholene (pH 3.5-10) (Pharmacia).
Protein concentration of all samples was adjusted to 2.2 mg/mL and 0.5 �L each sample was applied to each lane, lsoelectric point (pI) standard of pH 3.5 to pH 10 was applied with each sample so that a pi value could be assigned to selected bands on the gel. After electrophoresis, the gel was fixed in 5% TCA for 30 min and stained with Coomassie blue R-250 for 2 h. Isoelectric point values of each protein were then estimated.20,21
Immunodiagnosis of Fasciola species
Patient sera: Blood samples were obtained from six cases of acute fascioliasis that showed a high indirect haemagglutination (IHA) titre (≥ 1 in 2560). Serum was separated and used in immunodiffusion analysis and immunoelectrophoresis.
Immunodiffusion analysis: Analysis was carried out in 1% Noble agar (Difco, Detroit, MI, USA) in 0.1 mol/L Tricine buffer (Bio-Rad; pH 8.6) containing 3% polyethylene glycol (6000 K), as described by Harrington et al.22
Immunoelectrophoresis: Electrophoresis was carried out at 0.8 V/mm for 2 h using 1% agarose (Bio-Rad) in 0.1 mol/L veronal buffer (pH 8.6), as described by Mayer and Walker.23
Results
Electrophoretic patterns of native proteins prepared from worms fractioned at low pH (acid-PAGE) and high pH (alkaline-PAGE) are shown in Figures 1 and 2, respectively. Acid-PAGE showed three major protein peptides for F. hepatica (Fh1, Fh2 and Fh3) and two major protein peptides for F. gigantica (Fg1 and Fg2), separated by the speed of migration. Minor peptides were also found in F. hepatica (Fh4 to Fh8) and F. gigantica (Fg8 and Fg9), and some protein peptides were common to both species.
Figure 3 shows SDS-PAGE electrophoretic patterns of F. hepatica and F. gigantica proteins according to their molecular weights. F. hepatica samples were characterised by eight major peptide bands, with molecular weights estimated at 48, 45, 43.5, 37, 33, 29, 27 and 25.5 kDa. In addition, several minor bands were present. F. gigantica had only five major bands, with molecular weights 57.6, 54, 48, 29 and 27 kDa.
Isoelectric focusing patterns of F. hepatica and F. gigantica body proteins are shown in Figure 4. Seventeen bands were demonstrated for F. hepatica and 22 bands for F. gigantica in the pH range of 3.5 to 10. F. hepatica protein bands were located between pI 4.6-7.2, and in F gigantica the range was pI 4.2-9.5.
Six patients diagnosed with acute fascioliasis were included in the study. Figure 5A shows the results of immunodiffusion. Serum samples from acute patients showed two dense precipitin lines between the F. hepatica antigen and the corresponding antiserum, while two light precipitin lines were observed in the case of F. gigantica antigen. Figure 5B shows the results of immunoelectrophoresis. Several precipitin arcs were seen with the F. hepatica antigen, three of which were more dense than the others (one towards the anode, one towards the cathode and the third one towards the well of application). Only two preciptin arcs were seen with the F. gigantica antigen, one of which was dense.
Discussion
In Egypt, buffalo, cattle, sheep and goats are infected with F. gigantica.24 However, Farag et al.25 and Mansour et al.26 have reported that most human fascioliasis is caused by F. hepatica, and interestingly Curry et al.27 found F. hepatica in Egyptian mummies. Therefore, the present study attempted to characterise Fasciola species in animals and humans using molecular and immunological techniques.
Acid-PAGE, alkaline-PAGE, SDS-PAGE and isoelectric focusing were considered to be the most reliable of such methods for the differentiation of the two species. When protein samples of each species were fractionated at high pH (alkaline-PAGE) a number of peptides were isolated, some of which were common to both species
Using SDS-PAGE, a number of peptides were separated from each species on the gel and they differed in their migration position and thus their molecular weights. In the present study, F. hepatica showed bands with molecular weights ranging from 25.5 to 48 kDa, while F. gigantica showed bands that ranged from 27 to 54 kDa.
Rivera Marrero et al.28 studied the SDS-PAGE electrophoretic pattern of the E/S product of F. hepatica and found fractions of 25 to 48 kDa, 12 to 14 KDa and 150 to 160 kDa. Sampaio Silva et al.29 identified 11 peptide bands in F. hepatica, with molecular weights ranging from 12.4 to 116 kDa. Maleewong et al.30 reported SDS-PAGE analysis of both crude and fractionated E/S product of F. gigantica that revealed three major bands with molecular weights ranging from 14.4 to 27 kDa.
In the present work IEF identified approximately 17 bands from F. hepatica and 22 bands from F. gigantica in the pH range from 3.5 to 10. All protein bands were located between pI 4.6-7.2 and pI 4.2-9.5 in F. hepatica and F. gigantica, respectively.
Automated IEF has been used to distinguish F. hepatica from F. gigantica species by Lee and Zimmerman.19 F hepatica body protein showed 15 dominant peaks in the pH range of 4.6 to 9.3, whereas F. gigantica showed 18 dominant peaks in the pH range of 4.6 to 9.2, the majority of which shared between the two species.
In the study reported here, gel diffusion and immunoelectrophoresis using crude adult worm antigens were used to diagnose human fascioliasis; however, the antiserum proved more specific for the F. hepatica antigen. The results were confirmed by immunoelectrophoresis, in which more precipitin arcs were seen with F. hepatica antigen than with F. gigantica antigen.
[Reference]
References
1 Hammond JA. Human infection with the liver fluke Faciola gigantica. Trans R Soc Trop Med Hyg 1974; 68: 253-4.
2 Kachintorn U, Atisook K, Tesjaroen S, Lertakyamanee N, Plengvanit U. Faciola gigantka: the second case of human infection in Thailand. J Med Assoc Thai 1988; 71: 451-5.
3 Tesana S, Pamarapa A, Sae Sio TO. Acute cholecystitis and Fasciola sp. infection in Thailand: report of two cases. Southeast Asian J Trop Med Public Health 1989; 20: 447-52.
4 World Health Organization. Control of foodborne trematode infections. World Health Organization Tech Rep Ser 1995; 849: 1-157.
5 Stunkard HW. Intraspecific variation in parasitic flatworms. Syst Zool 1957; 6: 7-18.
6 Sazanov AM. On the polytypic nature of species of Fasciola L., 1758. Tr Vses Inst Gel' raintol 1973; 20: 155-61 (in Russian, with English summary).
7 Kendall SB. Relationships between the species of Fasciola and their molluscan hosts. Adv Parasitol 1965; 3: 59-98.
8 Oshima T, Akahane H, Shimazu T. Patterns of the variation of the common liver fluke (Fasciola sp.) in Japan. 1. Variations in the sizes and shapes of the worms and eggs. Jpn J Parasitol 1968; 17: 97-105. (in Japanese, with English summary).
9 Chu JK, Kim YK. Taxonomical study on the Fasciolidae in Korea. Korean J Parasitol 1967; 5: 139-46 (in Korean, with English abstract).
10 Yoshimura K. Disc electrophoretic comparison between Schistosoma japonicum and S. mansoni adult worms. Jpn J Parasitol 1968; 17: 282-303.
11 Yoshimura K. Paragonimus: electrophoretic fractionation of whole body proteins as an aid in specific identification of a species from Sado Island, Japan. Exp Parasitol 1969; 25: 107-17.
12 Johnson MR, Hoberg EP. Differentiation of Moniezia expansa and Moniezia benedeni (Eucestoda: cyclophyllidea) by isoelectric focusing. Can J Zool 1989; 67: 1471-5.
13 Kumaratilake LM, Thompson, RCA, Dunsmore JD. Infra-specific variation in Echinococcus: a biochemical approach. Z Parasitenkd 1979; 60: 291-4.
14 Tenter AM, Johnson MR, Zimmerman GL. Differentiation of Sarcocystis species in European sheep by isoelectric focusing. Parasitol Res 1989; 76: 107-14.
15 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-75.
16 Hames BD, Rickwood D. In: Gel electrophoresis of proteins: a practical approach. London: TRL Publishing, 1990: 34-48.
17 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 277: 680-5.
18 Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. Biol Chem 1969; 244: 4406-12.
19 Lee CG, Zimmerman GL. Banding patterns of Fasciola gigantica and F. hepatica (Trematoda) by isoelectric focusing. J Parasitol 1993; 79: 120-3.
20 Olsson J, Axi�-Fredricksson UB, Degerman M, Olsson B. Fast horizontal electrophoresis. I. Iso-electric focusing and polyacrylamide gel electrophoresis using Phast System(TM). Electrophoresis 1988; 9: 16-22.
21 Lee CG, Zimmerman GL, Bishop JK. Host influence on the banding profiles of whole body protein and excretory-secretory product of Fasciola hepatica (Trematoda) by isoelectric focusing. Vet Parasitol 1992; 41: 57-68.
22 Harrington JC, Fenton JW, Pert JH. Polymer-induced precipitation of antigen-antibody complexes. Precipiplex reactions. Immunochemistry 1971; 8: 413-21.
23 Mayer RJ, Walker JH. Methods for testing and using antibodies. In: Immunochemical methods in cell and molecular biology, London: Academic Press, 1990: 224-8.
24 Halawani AA, Gindy M. A short account on the economic importance of Fasciola gigantica, its effect on livestock and the snail vector. J Egypt Med Ass 1957; 40: 547.
25 Farag HF, Barakat RMR, Ragab M, Omar E. A focus of human fascioliasis in the Nile delta. Egypt J Trop Med Hyg 1979; 82: 188-90.
26 Mansour NS, Youssef FG, Mikhail EM, Boctor FN. Use of partially purified F. gigantica worm antigen in the serological diagnosis of human facioliasis in Egypt. Am J Trop Med Hyg 1983; 32: 550-4.
27 Curry A, Anfield C, Tapp E. Electron microscopy of the Manchester mummies. In: David AR, ed. Manchester Museum Mummies Project. Multidisciplinary research on ancient Egyptian mummified remains. Manchester: Manchester Museum, 1979: 103-11.
28 Rivera Marrero CA, Santiago N, Hillyer GV. Evaluation of immunodiagnostic antigens in the excretory-secretory products of Fasciola hepatica. J Parasitol 1988; 74: 646-52.
29 Sampaio Silva ML, Correa da Costa JM, Viana da Costa AM et al. Antigenic components of excretory-secretory products of adult Faciola hepatica recognized in human infections. Am J Trop Med Hyg 1996; 54: 146-8.
30 Maleewong W, Wongkham C, Intapan PM, Pipitgool V. Fasciola gigantica-specific antigens. Purification by a continuous-elution method and its evaluation for the diagnosis of human fascioliasis. Am J Trop Med Hyg 1999; 61: 648-51.
[Author Affiliation]
AMAL F ALLAM1, EL SAYED I EL-AGAMY2 and MADIHA H HELMY3
1 Parasitology Department, Medical Research Institute; 2 Dairy Science Department, Faculty of Agriculture; and 3 Biochemistry Department, Medical Research Institute Alexandria University, Alexandria, Egypt.
Accepted: 22 February 2002
[Author Affiliation]
Correspondence to: Dr Amal F Allam, Parasitology Department, Medical Research Institute, Alexandria University, 165 El Horreya Avenue, El Hadara, Alexandria, Egypt.
Email: sscbc@dataxprs.com.eg
