Parasitology Research

, Volume 103, Issue 1, pp 141–150 | Cite as

Early detection of Fasciola gigantica infection in buffaloes by enzyme-linked immunosorbent assay and dot enzyme-linked immunosorbent assay

  • Niranjan Kumar
  • S. GhoshEmail author
  • S. C. Gupta
Original Paper


In an attempt to develop a suitable serological test for early detection of Fasciola gigantica infection in buffaloes, a group of proteins were isolated from the somatic antigen of the parasite by immunoaffinity chromatography. The process of isolation of the proteins has been standardized and significant level of repeatability was achieved. To test the diagnostic potentiality of the antigens, two serological tests, viz., enzyme-linked immunosorbent assay (ELISA) and dot enzyme-linked immunosorbent assay, were standardized using the sera from experimentally noninfected (group A) and infected (group B) animals. Further, the sensitivity and the specificity of the tests were evaluated employing the field sera from animals of different parasitic load viz., F. gigantica positive (group C), F. gigantica and Gastrothylax crumenifer positive (group D), F. gigantica and Gigantocotyle explanatum positive (group E), a group of sera without F. gigantica but other trematode infection (group F), only G. crumenifer positive (group G), only G. explanatum positive (group H), G. crumenifer and G. explanatum positive (group I), and PM negative (group J) collected from slaughterhouses of Bareilly (Uttar Pradesh, India) and Patna (Bihar, India). In plate ELISA, the sensitivity of the antigen and the test was 75.75% while the specificity was 97%, 95%, and 98%, respectively, against G. crumenifer, G. explanatum, and mixed infection of G. crumenifer and G. explanatum, respectively. In the case of dot ELISA the sensitivity was 86.5% and specificity was 92.3%, 94.7%, and 90%, respectively, against G. crumenifer, G. explanatum, and mixed infection of G. crumenifer and G. explanatum, respectively. The potentiality of the antigen in the diagnosis of field infection is discussed.


Serum Number Parasitic Load Indirect ELISA Somatic Antigen Trematode Infection 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Sincere thanks are due to Indian Council of Agricultural Research, New Delhi, India for providing Research Fellowship to the senior author. Authors are grateful to the Director of the institute for providing necessary facilities.


  1. Aiken A, Learmoth M (1996) Protein determination by UV absorption. In: Walker JM (ed) The protein protocols hand book. Humana, TotowaGoogle Scholar
  2. Anderson N, Luong TT, Vo NG, Bui KL, Smooker PM, Spithill TW (1999) The sensitivity and specificity of two methods for detecting Fasciola infections in cattle. Vet Parasitol 83:15–24PubMedCrossRefGoogle Scholar
  3. Chowdhuri N (1994) Helminths of domesticated animals in Indian subcontinent. In: Chowdhuri N, Tada I (eds) Helminthology. Narosa, New Delhi, pp 73–114Google Scholar
  4. Cornelissen JB, Gaasenbeek CP, Borgsteede FH, Holland WG, Harmsen MM, Boersma WJ (2001) Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease. Int J Parasitol 31:728–737PubMedCrossRefGoogle Scholar
  5. Dixit AK, Yadav SC, Sharma RL (2002) 28 kDa Fasciola gigantica cysteine proteinase in the diagnosis of prepatent ovine fasciolosis. Vet Parasitol 109:233–247PubMedCrossRefGoogle Scholar
  6. Dixit AK, Yadav SC, Sharma RL (2004) Experimental bubaline fasciolosis: kinetics of antibody response using 28 kDa Fasciola gigantica cysteine proteinase as antigen. Trop Anim Hlth Prod 36:49–54CrossRefGoogle Scholar
  7. Doyle JJ (1973) Hypersensitivity reaction induced in calves by experimental infections with Fasciola hepatica. Int Arch Allergy Applied Immunol 45:752Google Scholar
  8. El-Kerdany ED, Abd-Alla NM, Sharaki OA (2002) Recognition of antigenic components of Fasciola gigantica and their use in immunodiagnosis. J Egypt Soc Parasitol 32:675–679PubMedGoogle Scholar
  9. Engvall E, Perlman P (1971) Enzyme linked immunosorbent assay (ELISA). Quantitative assay of IgG. Immunochem 8:871–874CrossRefGoogle Scholar
  10. Fagbemi BO, Guobadia EE (1995) Immunodiagnosis of fasciolosis in ruminants using a 28-kDa cysteine protease of Fasciola gigantica adult worms. Vet Parasitol 57:309–318PubMedCrossRefGoogle Scholar
  11. Fagbemi BO, Obarisiagbon IO, Mbuh JV (1995) Detection of circulating antigen in sera of Fasciola gigantica infected cattle with antibodies reactive with a Fasciola specific 88 kDa antigen. Vet Parasitol 58:235–246PubMedCrossRefGoogle Scholar
  12. Fagbemi BO, Aderibigbe OA, Guobadia EE (1997) The use of monoclonal antibody for the immunodiagnosis of Fasciola gigantica infection in cattle. Vet Parasitol 69:231–240PubMedCrossRefGoogle Scholar
  13. Fey H, Pfister H, Messerli J, Sturzenegger N, Grolimund F (1976) Methods of isolation, purification and quantitation of bovine immunoglobulins: a technical review. Zent Fur Vet Med 23:269Google Scholar
  14. Ghosh S, Rawat P, Velusamy R, Joseph D, Gupta SC, Singh BP (2005a) 27 kDa Fasciola gigantica glycoprotein for the diagnosis of prepatent fasciolosis in cattle. Vet Res Commun 29:123–135PubMedCrossRefGoogle Scholar
  15. Ghosh S, Rawat P, Gupta SC, Singh BP (2005b) Comparative diagnostic potentiality of ELISA and dot-ELISA in prepatent diagnosis of experimental Fasciola gigantica infection in cattle. Indian J Exp Biol 43:536–541PubMedGoogle Scholar
  16. Gupta SC (2001) Detection of natural bubaline fasciolosis by enzyme-linked immunosorbent assay using affinity purified Fasciola gigantica functional antigen. Indian J Anim Sci 16(1):99–102Google Scholar
  17. Harlow E, Lane D (1988) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  18. Hillyer G V, Soler de Galanes M, Buchon P, Bjorland J (1996) Herd evaluation by enzyme-linked immunosorbent assay for the determination of Fasciola hepatica infection in sheep and cattle from the Altiplano of Bolivia. Vet Parasitol 61:211–220PubMedCrossRefGoogle Scholar
  19. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:630–684CrossRefGoogle Scholar
  20. Linh BK, Thuy DT, My LN, Sasaki O, Yoshihara S (2003) Application of agar gel diffusion test to the diagnosis of fasciolosis in cattle and buffaloes in the red river delta of Vietnam. JARQ 37(3):201–205Google Scholar
  21. Lowry OH, Rosenbrough NJ, Farr AL, Randael RJ (1951) Protein measurement with the folin phenol reagent. J Biologic Chemist 193:265–275Google Scholar
  22. Mandal S, Yadav SC, Sharma RL (1998) Evaluation of Fasciola gigantica antigenic preparations in serodiagnosis of fasciolosis in sheep. J Parasitic Dis 22:25–29Google Scholar
  23. Maisonnave J (1999) Standardization of dot immunoperoxidase assay for field diagnosis of Fasciola hepatica infected cattle. Vet Parasitol 85:259–268PubMedCrossRefGoogle Scholar
  24. Ouchterlony O (1964) Gel diffusion technique. In: Ackroyd JF (ed) Immunological methods. Blackwell Scientific, Oxford, pp 55–78Google Scholar
  25. Pappas MG, Hajkowski R, Hockmeyer WT (1983) Dot-enzyme linked immunosorbent assay (dot-ELISA): a micro technique for the rapid diagnosis of visceral leishmaniasis. J Immunol Methods 64:205–214PubMedCrossRefGoogle Scholar
  26. Raina OK, Yadav SC, Sriveny D, Gupta SC (2006) Immunodiagnosis of bubaline fasciolosis with Fasciola gigantica cathepsin-L and recombinant cathepsin L 1-D protease. Acta Trop 98:145–151PubMedCrossRefGoogle Scholar
  27. Reichel MP (2002) Performance characteristics of an enzyme-linked immunosorbent assay for the detection of liver fluke (Fasciola hepatica) infection in sheep and cattle. Vet Parasitol 107:65–72PubMedCrossRefGoogle Scholar
  28. Rokni MB, Massoud J, Hanila A (2003) Comparison of adult somatic and cysteine proteinase antigens of Fasciola gigantica and their use in enzyme linked immunosorbent assay for serodiagnosis of human fasciolosis. Acta Trop 88:69–75PubMedCrossRefGoogle Scholar
  29. Sanchez-Andrade R, Paz-Silva A, Suarez J, Panadero R, Diez- Banos P, Morrondo P (2000) Use of sandwich enzyme-linked immunosorbent assay (SEA) for the diagnosis of natural Fasciola hepatica infection in cattle from Galicia (NW Spain). Vet Parasitol 93:39–46PubMedCrossRefGoogle Scholar
  30. Santiago N, Hillyer GV (1988) Antibody profile by EITB and ELISA of cattle and sheep infected with Fasciola hepatica. J Parasitol 74:810–818PubMedCrossRefGoogle Scholar
  31. Spithill TW, Piedrafita D, Smooker M (1997) Immunological approaches for the control of fasciolosis. Int J Parasitol 27:1221–1235PubMedCrossRefGoogle Scholar
  32. Talwar GP, Gupta SK (1992) Hand book of practical and clinical immunology. CBS, New DelhiGoogle Scholar
  33. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:607–610CrossRefGoogle Scholar
  34. Velusamy R, Singh BP, Sharma RL, Chandra D (2004) Detection of circulating 54 kDa antigen in sera of bovine calves experimentally infected with Fasciola gigantica. Vet Parasitol 119:187–195PubMedCrossRefGoogle Scholar
  35. Willadsen P, Riding GA, McKenna RV, Kemp DH, Tellam RL, Neilsen JN, Lahnstein J, Cobon GS, Gough JM (1989) Immunological control of parasitic arthropod. Identification of protective antigen from Boophilus microplus. J Immunol 143:1346–1351PubMedGoogle Scholar
  36. Yadav SC, Gupta SC (1993) dot-ELISA and DID in the diagnosis of Fasciola gigantica infection in rabbits. J Vet Parasitol 7:51–54Google Scholar
  37. Yadav SC, Saini M, Raina OK, Nambi PA, Jadav K, Sriveny D (2005) Fasciola gigantica cathepsin-L cysteine proteinase in the detection of early experimental fasciolosis in ruminants. Parasitol Res 97:527–534PubMedCrossRefGoogle Scholar
  38. Yokananth S, Ghosh S, Gupta SC, Suresh MG, Saravanan D (2005) Characterization of specific and cross-reacting antigens of Fasciola gigantica by immunoblotting. Parasitol Res 97:41–48PubMedCrossRefGoogle Scholar
  39. Zimmerman GL, Nelson MJ, Clark CRB (1985) Diagnosis of ovine fascioliasis by a dot-enzyme linked immunosorbent assay: a rapid diagnostic technique. Am J Vet Res 46(7):1513–1515PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Division of ParasitologyIndian Veterinary Research InstituteBareillyIndia

Personalised recommendations