Use of cloned excretory/secretory low-molecular-weight proteins of Cooperia oncophora in a serological assay.
نویسندگان
چکیده
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.
منابع مشابه
بررسی نقش نانوذره کیتوزان (Chitosan) همراه شده با پروتئینهای ترشحی-تراوشی لیشمانیا ماژور بر میزان اپوپتوز ماکروفاژهای موشی حساس شده با انگل
Background and Objective: Leishmaniasis is a public health problem caused by different types of Leishmania. The induction of apoptosis in infected macrophages is one of the main mechanisms for the escape of the parasite from the immune system. Preventing apoptosis and enhancing the ability to kill macrophages can be effective in treating or controlling leishmaniasis. In this study, the effect ...
متن کاملDetermination of somatic and excretory-secretory antigens of Fasciola hepatica and Fasciola gigantica using SDS-PAGE
Human fascioliasis due to unknown species and animal fascioliasis caused by both or one of Fasciola spp. are commonly seen in Iran. To compare electrophoretic patterns of somatic and excretory-secretory antigens of F. hepatica and F. gigantica by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the adult flukes were collected from infected slaughtered bovine livers. E/S a...
متن کاملIsolation and identification of excretory-secretory and somatic antigens from the Oestrus ovis larvae by SDS-PAGE and immunoblotting
Oestrus ovis is an economically important parasite of small ruminants and a zoonotic parasite with many reports of ophthalmomyiasis in human from Iran and other countries. The aim of the peresent studywas the isolation and identification of excretory-secretory (ES) and somatic (S) antigens of O. ovis second and third stage larvae (L2, L3) collected from Arabi sheep breeds loca...
متن کاملGenetic variability of glutamate-gated chloride channel genes in ivermectin-susceptible and -resistant strains of Cooperia oncophora.
The glutamate-gated chloride channels (GluCls) are members of the ligand-gated ion channel superfamily that are thought to be involved in the mode of action of ivermectin and mechanism of resistance. Using reverse-transcriptase PCR techniques, 2 full-length GluCl cDNAs, encoding GluClalpha3 and GluClbeta subunits, were cloned from Cooperia oncophora, a nematode parasite of cattle. The two seque...
متن کاملIdentification of excretory-secretory products from larval stages of Ostertagia circumcincta cultured in vitro by SDS-PAGE and immunoblotting
Exsheathing fluid (EF) and excretory-secretory products (ES) of infective third-stage cultured larvae ofOstertagia circumcincta were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Five and seven predominant proteins were found in the EF and ES products, respectively. Immunoblotting by sheep pre-infection serum did not react with any of the EF and ES proteins, b...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Journal of clinical microbiology
دوره 35 7 شماره
صفحات -
تاریخ انتشار 1997