title: Use of cloned excretory/secretory low-molecular-weight proteins of Cooperia oncophora in a serological assay.
abstract: The potential of Cooperia oncophora excretory/secretory (ES) proteins as antigens in a serological assay which aims to establish exposure levels in cattle was assessed. ES proteins were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis and immunoblotting. The N-terminal domains of two ES proteins were sequenced, and the corresponding cDNAs were cloned. Two cDNAs, designated CoES14.0 and CoES14.2, were expressed in Escherichia coli. The recombinant proteins were tested in an indirect enzyme-linked immunosorbent assay (ELISA) in which crude worm antigen (CWA) was used as a reference standard. In total, 67 reference serum samples were used: 27 negative serum samples, 29 C. oncophora-specific serum samples, 7 Dictyocaulus viviparus-specific serum samples, and 4 Ostertagia ostertagi-specific serum samples. This showed respective sensitivities and specificities of 17 and 84%, 0 and 100%, and 100 and 100% by the ELISAs with the three different types of proteins (CWA, CoES14.0, and CoES14.2, respectively). Since the CoES14.2 ELISA had the best sensitivity and specificity with reference sera, its specificity was further validated in an antigen inhibition ELISA. In this assay CoES14.2 and CWA preparations of C. oncophora, Cooperia curticei, O. ostertagi, Nematodirus helvetianus, Fasciola hepatica, D. viviparus, Haemonchus placei, and Trichostrongylus colubriformus were used as competitor antigens. This experiment showed that only the homologous antigens C. oncophora CWA and CoEs14.2 resulted in 100% inhibition. The CWA preparations of all other nematodes did not affect the ELISA, even if concentrations of 250 times the 50% inhibitory concentration of C. oncophora CWA were used. These results indicate that CoES14.2 does not share cross-reactive epitopes with heterologous CWAs. Finally, we tested the CoES14.2 ELISA with sequential serum samples from naturally infected groups of animals. The optical density values that were obtained correlated well with exposure levels based on cumulative egg excretion. Thus, the CoES14.2 ELISA seems to be a very sensitive tool for estimating exposure levels in cattle.
authors.affiliation: Institute of Infectious Diseases and Immunology, Department of Parasitology and Tropical Veterinary Medicine, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Poot
authors.forename: F N
authors.initials: FN
authors.lastname: Kooyman
authors.forename: P Y
authors.initials: PY
authors.lastname: Dop
authors.forename: H D
authors.initials: HD
authors.lastname: Schallig
authors.forename: M
authors.initials: M
authors.lastname: Eysker
authors.forename: A W
authors.initials: AW
authors.lastname: Cornelissen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009348:Nematoda / Q000276:immunology / Q000302:isolation & purification*; D010274:Parasitology / Q000379:methods*
chemical_list: D000947:Antigens, Helminth