Mechanical isolation and characterization of antigensfrom adult Anguillicola crassus

The serum Ig response of the European eel (Anguilla anguilla) to the bloodfeeding nematode Anguillicola crassus was studied. When tested by ELISA in a previous study, it was found necessary to purify the adult worm antigens in order to reduce the unwanted high background values. In the present study an alternative method is described and evaluated. Instead of using biochemical methods, adult worms were simply dissected and antigens prepared from the cuticle, female and male gonads, intestinal wall and the intestinal contents. The specific antibody levels in pooled sera from eels that were (1) non-infected; (2) naturally infected and (3) immunized with whole-worm extract, were tested by ELISA with these antigens and compared with the reaction to adult whole-worm homogenate (AWWH) and excretory-secretory (ES) antigens. In addition, the antigen preparations were characterized by sodium dodecyl polyacrylamide-gel electrophoresis (SDS-PAGE). The Western blotting technique (WB) was used to determine their recognition by antibodies in eel sera. The results obtained support the idea that remnants of eel blood present in the gastrointestinal tract of the adult worm generate high background values in the ELISA. However, when we characterized the antigen preparations that reacted similarly with low background values in the ELISA (i.e. the cuticle and the gonads), major differences were seen both in the protein banding pattern and between the bands that were recognized by the antibodies following WB. For example, the cuticle contained a distinct specific band with an approximate molecular weight of 45 kDa that was not seen in preparations of the gonads. It can be concluded that the cuticle of A. crassus contains specific antigens which can easily be isolated. The described method for antigen processing is simple and the isolated cuticle antigens proved to be specific in an ELISA measuring the antibody response to A. crassus in the European eel.