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.
A peptide-based indirect ELISA to detect cattle antibodies against Fasciola hepatica was developed and evaluated for its sensitivity and specificity. An immunogenic antigen released in vitro by F. hepatica was purified. After purification the sequence of the first 20 N-terminal aa of this protein showed considerable homology with cathepsin L-like proteinase. Based on its homology with cathepsin-L1, we further focused on this protein for diagnostic purpose. Predicted B-cell epitopes of cathepsin-L1 were synthesised as single synthetic peptides and tested with respect to their diagnostic potential. An indirect ELISA based on one of these peptides was (i) evaluated further and (ii) compared to the potential of an indirect ELISA with excretion/secretion antigens from adult F. hepatica, or (iii) purified cathepsin-L1. Specificity and sensitivity of the three ELISAs were assessed using sera from calves experimentally infected with pure isolates of Dictyocaulus viviparus, Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Schistosoma mattheei, Ascaris suum, Taenia saginata or F. hepatica, respectively, and sera from parasite-naive calves. In addition, sera were analysed from calves naturally infected with F. hepatica. The sensitivities of all three ELISAs were also very high, 98.9% (i), 100% (ii) and 100% (iii). The specificity of the peptide ELISA was very high, 99.8%, whereas specificities of the ES antigens and cathepsin-L1 ELISAs were only 82.8% and 94.6%. In experimentally infected cattle, F. hepatica-specific antibodies were first detected between days 21 and 28 p.i. with all three ELISAs, and the antibody levels persisted in the peptide ELISA until day 183 p.i. All sera from naturally infected calves were positive in the peptide ELISA. These results demonstrate that the peptide-based F. hepatica ELISA is a useful method for detecting antibodies in the sera from cattle infected with F. hepatica. This type of immunodiagnostic will therefore contribute to more accurate diagnosis and to timely curative treatment of animals.
DLO-Institute for Animal Science and Health, Department of Immunology, Pathobiology and Epidemiology, Lelystad, The Netherlands. j.b.w.j.cornelissen@id.dlo.nl
J B
JB
Cornelissen
C P
CP
Gaasenbeek
W
W
Boersma
F H
FH
Borgsteede
F J
FJ
van Milligen
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D000918:Antibody Specificity / Q000276:immunology; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics / Q000276:immunology*; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000276:immunology / Q000469:parasitology*; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D000939:Epitopes / Q000276:immunology*; D005210:Fasciola hepatica / Q000276:immunology*; D005211:Fascioliasis / Q000175:diagnosis / Q000662:veterinary*; D015801:Helminth Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013997:Time Factors
D000909:Antibodies, Helminth; D000939:Epitopes; D015801:Helminth Proteins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L