Parasitology Research

, Volume 117, Issue 5, pp 1521–1534 | Cite as

Comparison of recombinant cathepsins L1, L2, and L5 as ELISA targets for serodiagnosis of bovine and ovine fascioliasis

  • Victoria Martínez-Sernández
  • María J. Perteguer
  • Ana Hernández-González
  • Mercedes Mezo
  • Marta González-Warleta
  • Ricardo A. Orbegozo-Medina
  • Fernanda Romarís
  • Esperanza Paniagua
  • Teresa Gárate
  • Florencio M. Ubeira
Original Paper


Infections caused by Fasciola hepatica are of great importance in the veterinary field, as they cause important economic losses to livestock producers. Serodiagnostic methods, typically ELISA (with either native or recombinant antigens), are often used for early diagnosis. The use of native antigens, as in the MM3-SERO ELISA (commercialized as BIO K 211, BIO-X Diagnostics), continues to be beneficial in terms of sensitivity and specificity; however, there is interest in developing ELISA tests based on recombinant antigens to avoid the need to culture parasites. Of the antigens secreted by adult flukes, recombinant procathepsin L1 (rFhpCL1) is the most commonly tested in ELISA to date. However, although adult flukes produce three different clades of CLs (FhCL1, FhCL2, and FhCL5), to our knowledge, the diagnostic value of recombinant FhCL2 and FhCL5 has not yet been investigated. In the present study, we developed and tested three indirect ELISAs using rFhpCL1, rFhpCL2, and rFhpCL5 and evaluated their recognition by sera from sheep and cattle naturally infected with F. hepatica. Although the overall antibody response to these three rFhpCLs was similar, some animals displayed preferential recognition for particular rFhpCLs. Moreover, for cattle sera, the highest sensitivity was obtained using rFhpCL2 (97%), being equal for both rFhpCL1 and rFhpCL5 (87.9%), after adjusting cut-offs for maximum specificity. By contrast, for sheep sera, the sensitivity was 100% for the three rFhpCLs. Finally, the presence of truncated and/or partially unfolded molecules in antigen preparations is postulated as a possible source of cross-reactivity.


Cathepsin Fasciola hepatica Fascioliasis ELISA Cross-reactivity Serodiagnosis 



This work was supported by the Ministerio de Economía y Competitividad (Spain) [grant number AGL2011-30563-C03], Ministerio de Economía, Industria y Competitividad (INIA, Spain) [grants numbers RTA2017-00010-C02-01 and RTA2017-00010-C02-02], ISCIII-AESI (Spain) [project MPY 1279/15], Instituto de Salud Carlos III (Spain) [agreement PI14CIII/00076], and the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia, Spain) [grant number ED431B 2017/18]. VMS holds a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (Programa de Formación del Profesorado Universitario). RAOM holds a predoctoral fellowship from the Spanish Ministerio de Economía y Competitividad (Programa de Formación de Personal Investigador). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Blood and fecal samples were collected from naturally infected sheep and cattle by veterinarians from the “Centro de Investigaciones Agrarias de Mabegondo” (INGACAL, A Coruña, Spain). The samples were collected either during routine control and treatment of herds (sheep) or in the slaughterhouse (cattle). All procedures were carried out in strict accordance with Spanish and EU legislation (Law 32/2007, R.D. 53/2013, and Council Directive 2010/63/EU).

Conflict of interests

The authors declare that they have no competing interests.


  1. Abdel-Rahman SM, O'Reilly KL, Malone JB (1998) Evaluation of a diagnostic monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of a 26- to 28-kd Fasciola hepatica coproantigen in cattle. Am J Vet Res 59:533–537PubMedGoogle Scholar
  2. Anadón AM, Rodríguez E, Gárate MT, Cuéllar C, Romarís F, Chivato T, Rodero M, González-Díaz H, Ubeira FM (2010) Diagnosing human anisakiasis: recombinant Ani s 1 and Ani s 7 allergens versus the UniCAP 100 fluorescence enzyme immunoassay. Clin Vaccine Immunol 17:496–502CrossRefPubMedPubMedCentralGoogle Scholar
  3. 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 83:15–24CrossRefPubMedGoogle Scholar
  4. Andrews SJ (1999) The life cycle of Fasciola hepatica. In: Dalton JP (ed) Fasciolosis. CABI Publishing, Wallingford, pp 1–29Google Scholar
  5. Audicana MT, Kennedy MW (2008) Anisakis simplex: from obscure infectious worm to inducer of immune hypersensitivity. Clin Microbiol Rev 21:360–379CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bloemhoff Y, Forbes A, Danaher M, Good B, Morgan E, Mulcahy G, Sekiya M, Sayers R (2015) Determining the prevalence and seasonality of Fasciola hepatica in pasture-based dairy herds in Ireland using a bulk tank milk ELISA. Ir Vet J 68:16CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bozas SE, Panaccio M, Creaney J, Dosen M, Parsons JC, Vlasuk GV, Walker ID, Spithill TW (1995) Characterisation of a novel Kunitz-type molecule from the trematode Fasciola hepatica. Mol Biochem Parasitol 74:19–29CrossRefPubMedGoogle Scholar
  8. Carnevale S, Rodríguez MI, Santillán G, Labbe JH, Cabrera MG, Bellegarde EJ, Velásquez JN, Trgovcic JE, Guarnera EA (2001a) Immunodiagnosis of human fascioliasis by an enzyme-linked immunosorbent assay (ELISA) and a micro-ELISA. Clin Diagn Lab Immunol 8:174–177PubMedPubMedCentralGoogle Scholar
  9. Carnevale S, Rodríguez MI, Guarnera EA, Carmona C, Tanos T, Angel SO (2001b) Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase. Diagn Microbiol Infect Dis 41:43–49CrossRefPubMedGoogle Scholar
  10. Charlier J, Duchateau L, Claerebout E, Williams D, Vercruysse J (2007) Associations between anti-Fasciola hepatica antibody levels in bulk-tank milk samples and production parameters in dairy herds. Prev Vet Med 78:57–66CrossRefPubMedGoogle Scholar
  11. Charlier J, De Meulemeester L, Claerebout E, Williams D, Vercruysse J (2008) Qualitative and quantitative evaluation of coprological and serological techniques for the diagnosis of fasciolosis in cattle. Vet Parasitol 153:44–51CrossRefPubMedGoogle Scholar
  12. Charlier J, De Cat A, Forbes A, Vercruysse J (2009) Measurement of antibodies to gastrointestinal nematodes and liver fluke in meat juice of beef cattle and associations with carcass parameters. Vet Parasitol 166:235–240CrossRefPubMedGoogle Scholar
  13. Charlier J, Vercruysse J, Morgan E, van Dijk J, Williams DJ (2014) Recent advances in the diagnosis, impact on production and prediction of Fasciola hepatica in cattle. Parasitology 141:326–335CrossRefPubMedGoogle Scholar
  14. Cornelissen JB, Gaasenbeek CP, Boersma 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 29:685–696CrossRefPubMedGoogle Scholar
  15. 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 31:728–737CrossRefPubMedGoogle Scholar
  16. Correa AC, Escobar JS, Durand P, Renaud F, David P, Jarne P, Pointier JP, Hurtrez-Bousses S (2010) Bridging gaps in the molecular phylogeny of the Lymnaeidae (Gastropoda: Pulmonata), vectors of Fascioliasis. BMC Evol Biol 10:381CrossRefPubMedPubMedCentralGoogle Scholar
  17. Cuéllar C, Daschner A, Valls A, De Frutos C, Fernández-Figares V, Anadón AM, Rodríguez E, Gárate T, Rodero M, Ubeira FM (2012) Ani s 1 and Ani s 7 recombinant allergens are able to differentiate distinct Anisakis simplex-associated allergic clinical disorders. Arch Dermatol Res 304:283–288CrossRefPubMedGoogle Scholar
  18. Cwiklinski K, Dalton JP, Dufresne PJ, La Course J, Williams DJ, Hodgkinson J, Paterson S (2015) The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution. Genome Biol 16:71CrossRefPubMedPubMedCentralGoogle Scholar
  19. DeLong ER, DeLong DM, Clarke-Pearson DL (1988) Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics 44:837–845CrossRefPubMedGoogle Scholar
  20. Di Maggio LS, Tirloni L, Pinto AF, Diedrich JK, Yates JR III, Benavides U, Carmona C, da Silva Vaz I Jr, Berasain P (2016) Across intra-mammalian stages of the liver fluke Fasciola hepatica: a proteomic study. Sci Rep 6:32796CrossRefPubMedPubMedCentralGoogle Scholar
  21. Dowd AJ, Tort J, Roche L, Ryan T, Dalton JP (1997) Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae. Mol Biochem Parasitol 88:163–174CrossRefPubMedGoogle Scholar
  22. Duscher R, Duscher G, Hofer J, Tichy A, Prosl H, Joachim A (2011) Fasciola hepatica - monitoring the milky way? The use of tank milk for liver fluke monitoring in dairy herds as base for treatment strategies. Vet Parasitol 178:273–278CrossRefPubMedGoogle Scholar
  23. Espino AM, Dumenigo BE (2003) Fasciola hepatica. In: Miliotis MD, Bier JW (eds) International handbook of foodborne pathogens. Marcel Dekker, Inc, New York, pp 539–562Google Scholar
  24. Espino AM, Finlay CM (1994) Sandwich enzyme-linked immunosorbent assay for detection of excretory secretory antigens in humans with fascioliasis. J Clin Microbiol 32:190–193PubMedPubMedCentralGoogle Scholar
  25. Espinoza JR, Maco V, Marcos L, Saez S, Neyra V, Terashima A, Samalvides F, Gotuzzo E, Chavarry E, Huaman MC, Bargues MD, Valero MA, Mas-Coma S (2007) Evaluation of Fas2-ELISA for the serological detection of Fasciola hepatica infection in humans. Am J Trop Med Hyg 76:977–982PubMedGoogle Scholar
  26. George SD, Vanhoff K, Baker K, Lake L, Rolfe PF, Seewald W, Emery DL (2017) Application of a coproantigen ELISA as an indicator of efficacy against multiple life stages Fasciola hepatica infections in sheep. Vet Parasitol 246:60–69CrossRefPubMedGoogle Scholar
  27. Gonzales Santana B, Dalton JP, Vásquez-Camargo F, Parkinson M, Ndao M (2013) The diagnosis of human fascioliasis by enzyme-linked immunosorbent assay (ELISA) using recombinant cathepsin L protease. PLoS Negl Trop Dis 7:e2414CrossRefPubMedPubMedCentralGoogle Scholar
  28. Gottstein B, Schneeberger M, Boubaker G, Merkle B, Huber C, Spiliotis M, Muller N, Gárate T, Doherr MG (2014) Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human fasciolosis. PLoS Negl Trop Dis 8:e2860CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kofta W, Mieszczanek J, Plucienniczak G, Wedrychowicz H (2000) Successful DNA immunisation of rats against fasciolosis. Vaccine 18:2985–2990CrossRefPubMedGoogle Scholar
  30. Kuerpick B, Schnieder T, Strube C (2013) Evaluation of a recombinant cathepsin L1 ELISA and comparison with the Pourquier and ES ELISA for the detection of antibodies against Fasciola hepatica. Vet Parasitol 193:206–213CrossRefPubMedGoogle Scholar
  31. Lilie H, Schwarz E, Rudolph R (1998) Advances in refolding of proteins produced in E. coli. Curr Opin Biotechnol 9:497–501CrossRefPubMedGoogle Scholar
  32. Lin AH, Nepstad I, Florvaag E, Egaas E, Van Do T (2014) An extended study of seroprevalence of anti-Anisakis simplex IgE antibodies in Norwegian blood donors. Scand J Immunol 79:61–67CrossRefPubMedGoogle Scholar
  33. MAFF (1971) Manual of veterinary parasitological laboratory techniques. HM Stationery Off, LondonGoogle Scholar
  34. Martínez-Sernández V, Muiño L, Perteguer MJ, Gárate T, Mezo M, González-Warleta M, Muro A, Correia da Costa JM, Romarís F, Ubeira FM (2011) Development and evaluation of a new lateral flow immunoassay for serodiagnosis of human fasciolosis. PLoS Negl Trop Dis 5:e1376CrossRefPubMedPubMedCentralGoogle Scholar
  35. Martínez-Sernández V, Mezo M, González-Warleta M, Perteguer MJ, Muiño L, Guitián E, Gárate T, Ubeira FM (2014) The MF6p/FhHDM-1 major antigen secreted by the trematode parasite Fasciola hepatica is a heme-binding protein. J Biol Chem 289:1441–1456CrossRefPubMedGoogle Scholar
  36. Martínez-Sernández V, Orbegozo-Medina RA, González-Warleta M, Mezo M, Ubeira FM (2016) Rapid enhanced MM3-COPRO ELISA for detection of Fasciola coproantigens. PLoS Negl Trop Dis 10:e0004872CrossRefPubMedPubMedCentralGoogle Scholar
  37. Mas-Coma S, Valero MA, Bargues MD (2009) Fasciola, lymnaeids and human fascioliasis, with a global overview on disease transmission, epidemiology, evolutionary genetics, molecular epidemiology and control. Adv Parasitol 69:41–146CrossRefPubMedGoogle Scholar
  38. Mas-Coma S, Agramunt VH, Valero MA (2013) Direct and indirect affection of the central nervous system by Fasciola infection. Handb Clin Neurol 114:297–310CrossRefPubMedGoogle Scholar
  39. Mas-Coma S, Bargues MD, Valero MA (2014) Diagnosis of human fascioliasis by stool and blood techniques: update for the present global scenario. Parasitology 141:1918–1946CrossRefPubMedGoogle Scholar
  40. Mezo M, González-Warleta M, Ubeira FM (2003) Optimized serodiagnosis of sheep fascioliasis by Fast-D protein liquid chromatography fractionation of Fasciola hepatica excretory-secretory antigens. J Parasitol 89:843–849CrossRefPubMedGoogle Scholar
  41. Mezo M, González-Warleta M, Carro C, Ubeira FM (2004) An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3). J Parasitol 90:845–852CrossRefPubMedGoogle Scholar
  42. Mezo M, González-Warleta M, Ubeira FM (2007) The use of MM3 monoclonal antibodies for the early immunodiagnosis of ovine fascioliasis. J Parasitol 93:65–72CrossRefPubMedGoogle Scholar
  43. Mezo M, González-Warleta M, Castro-Hermida JA, Carro C, Ubeira FM (2010a) Kinetics of anti-Fasciola IgG antibodies in serum and milk from dairy cows during lactation, and in serum from calves after feeding colostrum from infected dams. Vet Parasitol 168:36–44CrossRefPubMedGoogle Scholar
  44. Mezo M, González-Warleta M, Castro-Hermida JA, Muiño L, Ubeira FM (2010b) Field evaluation of the MM3-SERO ELISA for detection of anti-Fasciola IgG antibodies in milk samples from individual cows and bulk milk tanks. Parasitol Int 59:610–615CrossRefPubMedGoogle Scholar
  45. Mezo M, González-Warleta M, Castro-Hermida JA, Muiño L, Ubeira FM (2011) Association between anti-F. hepatica antibody levels in milk and production losses in dairy cows. Vet Parasitol 180:237–242CrossRefPubMedGoogle Scholar
  46. Muiño L, Perteguer MJ, Gárate T, Martínez-Sernández V, Beltrán A, Romarís F, Mezo M, González-Warleta M, Ubeira FM (2011) Molecular and immunological characterization of Fasciola antigens recognized by the MM3 monoclonal antibody. Mol Biochem Parasitol 179:80–90CrossRefPubMedGoogle Scholar
  47. Norbury LJ, Hung A, Beckham S, Pike RN, Spithill TW, Craik CS, Choe Y, Fecondo JV, Smooker PM (2012) Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1-P4 specificity reveals an unusual preference. Biochimie 94:1119–1127CrossRefPubMedGoogle Scholar
  48. O'Neill SM, Parkinson M, Dowd AJ, Strauss W, Angles R, Dalton JP (1999) Short report: Immunodiagnosis of human fascioliasis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase. Am J Trop Med Hyg 60:749–751CrossRefPubMedGoogle Scholar
  49. Rapsch C, Schweizer G, Grimm F, Kohler L, Bauer C, Deplazes P, Braun U, Torgerson PR (2006) Estimating the true prevalence of Fasciola hepatica in cattle slaughtered in Switzerland in the absence of an absolute diagnostic test. Int J Parasitol 36:1153–1158CrossRefPubMedGoogle Scholar
  50. Robichon C, Luo J, Causey TB, Benner JS, Samuelson JC (2011) Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography. Appl Environ Microbiol 77:4634–4646CrossRefPubMedPubMedCentralGoogle Scholar
  51. Robinson MW, Dalton JP (2009) Zoonotic helminth infections with particular emphasis on fasciolosis and other trematodiases. Philos Trans R Soc Lond Ser B Biol Sci 364:2763–2776CrossRefGoogle Scholar
  52. Robinson MW, Tort JF, Lowther J, Donnelly SM, Wong E, Xu W, Stack CM, Padula M, Herbert B, Dalton JP (2008) Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulence-associated factors. Mol Cell Proteomics 7:1111–1123CrossRefPubMedGoogle Scholar
  53. Robinson MW, Menon R, Donnelly SM, Dalton JP, Ranganathan S (2009) An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host. Mol Cell Proteomics 8:1891–1907CrossRefPubMedPubMedCentralGoogle Scholar
  54. Salimi-Bejestani MR, Daniel R, Cripps P, Felstead S, Williams DJ (2007) Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to Fasciola hepatica in milk. Vet Parasitol 149:290–293CrossRefPubMedGoogle Scholar
  55. Schweizer G, Braun U, Deplazes P, Torgerson PR (2005) Estimating the financial losses due to bovine fasciolosis in Switzerland. Vet Rec 157:188–193CrossRefPubMedGoogle Scholar
  56. Selemetas N, Phelan P, O'Kiely P, Waal T (2014) Weather and soil type affect incidence of fasciolosis in dairy cow herds. Vet Rec 175:371CrossRefPubMedGoogle Scholar
  57. Smith D, Tikhonova IG, Jewhurst HL, Drysdale OC, Dvorak J, Robinson MW, Cwiklinski K, Dalton JP (2016) Unexpected activity of a novel Kunitz-type inhibitor: inhibition of cysteine proteases but not serine proteases. J Biol Chem 291:19220–19234CrossRefPubMedPubMedCentralGoogle Scholar
  58. Smooker PM, Whisstock JC, Irving JA, Siyaguna S, Spithill TW, Pike RN (2000) A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica. Protein Sci 9:2567–2572CrossRefPubMedPubMedCentralGoogle Scholar
  59. Smooker PM, Jayaraj R, Pike RN, Spithill TW (2010) Cathepsin B proteases of flukes: the key to facilitating parasite control? Trends Parasitol 26:506–514CrossRefPubMedGoogle Scholar
  60. Ubeira FM, Muiño L, Valero MA, Periago MV, Pérez-Crespo I, Mezo M, González-Warleta M, Romarís F, Paniagua E, Cortizo S, Llovo J, Más-Coma S (2009) MM3-ELISA detection of Fasciola hepatica coproantigens in preserved human stool samples. Am J Trop Med Hyg 81:156–162PubMedGoogle Scholar
  61. Valero MA, Ubeira FM, Khoubbane M, Artigas P, Muiño L, Mezo M, Pérez-Crespo I, Periago MV, Mas-Coma S (2009) MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica. Vet Parasitol 159:77–81CrossRefPubMedGoogle Scholar
  62. Valero MA, Bargues MD, Khoubbane M, Artigas P, Quesada C, Berinde L, Ubeira FM, Mezo M, Hernández JL, Agramunt VH, Mas-Coma S (2016) Higher physiopathogenicity by Fasciola gigantica than by the genetically close F. hepatica: experimental long-term follow-up of biochemical markers. Trans R Soc Trop Med Hyg 110:55–66CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Victoria Martínez-Sernández
    • 1
  • María J. Perteguer
    • 2
  • Ana Hernández-González
    • 2
  • Mercedes Mezo
    • 3
  • Marta González-Warleta
    • 3
  • Ricardo A. Orbegozo-Medina
    • 1
  • Fernanda Romarís
    • 1
  • Esperanza Paniagua
    • 1
  • Teresa Gárate
    • 2
  • Florencio M. Ubeira
    • 1
  1. 1.Laboratorio de Parasitología, Facultad de FarmaciaUniversidad de Santiago de CompostelaSantiago de CompostelaSpain
  2. 2.Centro Nacional de MicrobiologíaInstituto de Salud Carlos IIIMajadahondaSpain
  3. 3.Laboratorio de Parasitología, Centro de Investigaciones Agrarias de MabegondoINGACALAbegondoSpain

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