Comparison of recombinant cathepsins L1, L2, and L5 as ELISA targets for serodiagnosis of bovine and ovine fascioliasis
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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.
KeywordsCathepsin Fasciola hepatica Fascioliasis ELISA Cross-reactivity Serodiagnosis
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.
- 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
- Andrews SJ (1999) The life cycle of Fasciola hepatica. In: Dalton JP (ed) Fasciolosis. CABI Publishing, Wallingford, pp 1–29Google Scholar
- 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
- 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
- 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
- MAFF (1971) Manual of veterinary parasitological laboratory techniques. HM Stationery Off, LondonGoogle Scholar
- 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
- 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
- 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
- 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
- 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
- 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