Skip to main content
SpringerLink
Log in

Isolation, identification and expression of a Fasciola hepatica cDNA encoding a 2.9-kDa recombinant protein for the diagnosis of ovine fasciolosis

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

A 400-bp Fasciola hepatica cDNA clone was isolated from an expression library by immunological screening using rat sera taken 2 weeks after experimental infection. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 78 bp which encoded a 25 amino acid polypeptide with a predicted molecular weight of 2.9 kDa. This polypeptide was expressed in bacteria as a GST-fusion protein and used for the production of specific antigen. The 2.9 kDa recombinant protein (APS) was evaluated against sera from experimentally infected sheep using an indirect ELISA, and the results were compared to those obtained using F. hepatica excretory/secretory products (ESP). The pattern of IgG was very similar both against the recombinant and the native proteins, increasing early following the infection. After treatment with triclabendazole, the IgG response against the APS seroreverted to negative values, whereas it remained elevated against the ESP. We conclude that this recombinant protein could be used in diagnostic assays for the identification of recently infected sheep.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Arriaga de Morilla C, Paniagua R, Ruíz Navarrete A, Bautista CR, Morilla A (1989) Comparison of dot enzyme linked immunosorbent assay (dot-ELISA), passive haemaglutination test (PHT) and thin layer immunoassay (TIA) in the diagnosis of natural or experimental F. hepatica infections in sheep. Vet Parasitol 30:197–203

    Article  PubMed  Google Scholar 

  • Carnevale S, Rodríguez M, Guarnera EA, Carmona C, Tanos T, Angel S (2001) Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase. Diagn Microbiol Infect Dis 41:43–49

    Article  CAS  PubMed  Google Scholar 

  • Casanueva R, Hillyer GV, Ramajo V, Oleaga A, Espinoza EY, Muro A (2001) Immunoprophylaxis against Fasciola hepatica in rabbits using a recombinant Fh15 fatty acid-binding protein. J Parasitol 87:697–700

    CAS  PubMed  Google Scholar 

  • Cornelissen JB, Gaasenbeek CP, Boersman W, Borgsteede FH, Van Milligen FJ (1999) Use of a pre-selected epitope of cathepsin-L1 in a highly specific peptide-based immunoassay for the diagnosis of Fasciola hepatica infections in cattle. Int J Parasitol 29:685–696

    Article  CAS  PubMed  Google Scholar 

  • 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 31:728–737

    Article  CAS  PubMed  Google Scholar 

  • Dalton JP, McGonigle S, Rolph TP, Andrews SJ (1996) Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinases and with hemoglobin. Infect Immun 64:5066–5074

    CAS  PubMed  Google Scholar 

  • Espino AM, Marcet R, Finlay CM (1997) Fasciola hepatica: detection of antigenemia and coproantigens in experimentally infected rats. Exp Parasitol 85:117–120

    Article  CAS  PubMed  Google Scholar 

  • Espino AM, Díaz A, Pérez A, Finlay CM (1998) Dynamics of antigenemia and coproantigens during a human Fasciola hepatica outbreak. J Clin Microbiol 36:2723–2726

    CAS  PubMed  Google Scholar 

  • Fairweather I, Boray JC (1999) Fasciolicides: efficacy, actions, resistance and its management. Vet J 158:81–112

    Article  CAS  PubMed  Google Scholar 

  • Hanna RE (1980) Fasciola hepatica: glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity. Exp Parasitol 50:103–114

    Article  CAS  PubMed  Google Scholar 

  • Kaplan RM (2001) Fasciola hepatica: a review of the economic impact in cattle and considerations for control. Vet Ther 2:40–50

    Google Scholar 

  • Keegan PS, Trudgett A (1992) Fasciola hepatica in the rat: immune responses associated with the development of resistance to infection. Parasitol Immunol 14:657–669

    Google Scholar 

  • Knox DP, Redmond DL, Skuce PJ, Newlands GF (2001) The contribution of molecular biology to the development of vaccines against nematode and trematode parasites of domestic ruminants. Vet Parasitol 101:311–335

    Article  CAS  PubMed  Google Scholar 

  • Levieux D, Levieux A, Mage C, Garel JP (1992) Immunological detection of chemotherapeutic success in bovine fasciolosis using the specific antigen f2. Vet Parasitol 45:81–88

    Article  CAS  PubMed  Google Scholar 

  • Muro A, Ramajo V, López J, Simón F, Hillyer GV (1997) Fasciola hepatica: vaccination of rabbits with native and recombinant antigens related to fatty acid binding proteins. Vet Parasitol 69:219–229

    Article  CAS  PubMed  Google Scholar 

  • O’Neill SM, Parkinson M, Strauss W, Angles R, Dalton JP (1998) Immunodiagnosis of Fasciola hepatica infection (fascioliasis) in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase. Am J Trop Med Hyg 58:417–423

    PubMed  Google Scholar 

  • O’Neill SM, Parkinson M, Dowd AJ, Strauss W, Angles R, Dalton JP (1999) Immunodiagnosis of human fascioliasis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase. Am J Trop Med Hyg 60:749–751

    PubMed  Google Scholar 

  • Paz A, Sánchez-Andrade R, Panadero R, Díez P, Morrondo P (1998) IgG isotype specific immune response in rats infected with Fasciola hepatica. Vet Parasitol 79:229–237

    Article  CAS  PubMed  Google Scholar 

  • Paz A, Sánchez-Andrade R, Panadero R, Suárez JL, Díez-Baños P, Morrondo P (1999) Subclass profile of specific IgG antibodies in rats challenged during acute and chronic primary infection with Fasciola hepatica. Parasitol Res 85:770–775

    Article  CAS  PubMed  Google Scholar 

  • Paz-Silva A, Sánchez-Andrade R, Suárez JL, Pedreira J, Arias M, López C, Panadero R, Díaz P, Díez-Baños P, Morrondo P (2003) Prevalence of natural ovine fasciolosis shown by demonstrating the presence of serum circulating antigens. Parasitol Res 91:328–331

    Article  CAS  PubMed  Google Scholar 

  • Prichard R (1997) Application of molecular biology in veterinary parasitology. Vet Parasitol 71:155–175

    Article  CAS  PubMed  Google Scholar 

  • Rodríguez-Pérez J, Hillyer GV (1995) Detection of excretory-secretory circulating antigens in sheep infected with Fasciola hepatica and with Schistosoma mansoni and Fasciola hepatica. Vet Parasitol 56:57–66

    Article  PubMed  Google Scholar 

  • Rodríguez-Pérez J, Rodríguez-Medina JR, García-Blanco MA, Hillyer GV (1992) Fasciola hepatica: molecular cloning, nucleotide sequence, and expression of a gene encoding a polypeptide homologous to a Schistosoma mansoni fatty acid-binding protein. Exp Parasitol 74:400–407

    Article  PubMed  Google Scholar 

  • Sánchez-Andrade R, Paz-Silva A, Suárez J, Panadero R, Díez-Baños P, Morrondo P (2000) Use of a sandwich-enzyme-linked immunosorbent assay (SEA) for the diagnosis of natural Fasciola hepatica infection in cattle from Galicia (NW Spain). Vet Parasitol 93:39–46

    Article  PubMed  Google Scholar 

  • Sánchez-Andrade R, Paz-Silva A, Suárez JL, Panadero R, Pedreira J, Díez-Baños P, Morrondo P (2001) Effect of fasciolicides on the antigenaemia in sheep naturally infected with Fasciola hepatica. Parasitol Res 87:609–614

    Article  PubMed  Google Scholar 

  • Sexton JL, Wilce MC, Colin T, Wijffels GL, Salvatore L, Feil S, Parker MW, Spithill TW (1994) Vaccination of sheep against Fasciola hepatica with glutathione S-transferase. Identification and mapping of antibody epitopes on a three-dimensional model of the antigen. J Immunol 152:1861–1872

    CAS  PubMed  Google Scholar 

  • Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31–40

    Article  CAS  PubMed  Google Scholar 

  • Smooker PM, Steeper KR, Drew DR, Strugnell RA, Spithill TW (1999) Humoral responses in mice following vaccination with DNA encoding glutathione S-transferase of Fasciola hepatica: effects of mode of vaccination and the cellular compartment of antigen expression. Parasite Immunol 21:357–364

    Article  CAS  PubMed  Google Scholar 

  • Spithill TW, Dalton JP (1998) Progress in development of liver fluke vaccines. Parasitol Today 14:224–228

    Article  Google Scholar 

  • Trudgett A, Anderson A, Hanna RE (1988) Use of immunosorbent-purified antigens of Fasciola hepatica in enzyme immunoassays. Res Vet Sci 44:262–263

    CAS  PubMed  Google Scholar 

  • Trudgett A, McNair AT, Hoey EM, Keegan PS, Dalton JP, Rima BK, Miller A, Ramasamy P (2000) The major tegumental antigen of Fasciola hepatica contains repeated elements. Parasitology 121:185–191

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported, in part, by a research grant from the Xunta de Galicia (XUGA, Spain) to Dr. A. Paz-Silva, and by the Projects XUGA PGIDT00PXI26102PR (Xunta de Galicia, Spain), DCICYT AGL2001-2990 (Ministerio de Ciencia y Tecnología, Spain), NIH-NIGMS Grant No. SO6 GM08224, NCRR-RCMI G12 RR03051 (USA), and NSF-EPSCOR SPACS Program (USA), and complies with the current laws for Animal Health Research in Spain. We are indebted to Mrs. B. Valcárcel for preparing and editing the manuscript. The nucleotide sequence data reported in this paper have been submitted to GenBank database with accession no. AY676331.

Author information

Authors and Affiliations

  1. Departamento de Patología Animal, Parasitología y Enfermedades Parasitarias y Epidemiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27071 , Lugo, Spain

    A. Paz-Silva, R. Sánchez-Andrade, M. Arias, P. Morrondo & P. Díez-Baños

  2. Laboratory of Parasite Immunology and Pathology, School of Medicine, University of Puerto Rico, Suite 617-A, Medical Sciences Campus, 00936–5067, San Juan, Puerto Rico

    G. V. Hillyer

  3. Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, 00936–5067, San Juan, Puerto Rico

    J. R. Rodríguez-Medina

Authors
  1. A. Paz-Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G. V. Hillyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R. Sánchez-Andrade
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. R. Rodríguez-Medina
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. Arias
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. P. Morrondo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. P. Díez-Baños
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. Paz-Silva.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Paz-Silva, A., Hillyer, G.V., Sánchez-Andrade, R. et al. Isolation, identification and expression of a Fasciola hepatica cDNA encoding a 2.9-kDa recombinant protein for the diagnosis of ovine fasciolosis. Parasitol Res 95, 129–135 (2005). https://doi.org/10.1007/s00436-004-1202-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-004-1202-9

Keywords

Search

Navigation