Evaluation of an ELISA and immunoblotting for studying the humoral immune response in Anguillicola crassus infected European eel Anguilla anguilla.

The applicability of an enzyme-linked immunosorbent assay (ELISA) for the detection of anguillicolosis in feral eels was examined using a crude antigen preparation from the body wall of adult Anguillicola crassus. The screening consisted of samples from 100 feral European eels Anguilla anguilla. As a reference the actual status of infection was determined by dissection of the eels' swim-bladders. The ELISA results were compared with a background value calculated from the results obtained from 43 non-infected farm eels. The screened samples had a high prevalence of A. crassus (83 %); however, the specificity and the negative predictive value of the ELISA were low compared to the high positive predictive value. Nonetheless, the reproducibility (precision) of the test was satisfactory, and for the non-infected reference group specificity was 97.7 %. Although the ELISA, as used in the present study, is not applicable for diagnostic purposes, it represents a useful tool for the investigation of the specific humoral immune response of eels against A. crassus under controlled experimental conditions. Immunoblots using crude antigen preparations from different parts of adult A. crassus as well as a crude somatic third-stage (L3) antigen preparation illustrated that only antigens associated with the body wall of adult A. crassus are potentially suitable for diagnostic purposes. Despite the fact that antibodies against Raphidascaris acus cross-reacted with 3 body wall antigens of A. crassus, the most encouraging results were obtained with the antigen preparation from the outer cuticle of adult A. crassus which yielded a conspicuous, broad band at about 100 kDa.

Humoral immune response of European eel Anguilla anguilla experimentally infected with Anguillicola crassus.

A humoral immune response of the European eel Anguilla anguilla elicited by an experimental infection was demonstrated for the first time against the swimbladder nematode Anguillicola crassus. Eels were experimentally infected once or repeatedly and the antibody response was observed over a period of 325 d. Specific antibodies against A. crassus in the peripheral blood of the eels were measured using an ELISA and the immunoblot technique. Anti-A. crassus antibodies were first observed 8 wk post infection, and appeared to be independent of both the number of infective third stage larvae (L3) administered and the frequency of administration. However, individual eels showed great differences in the course of the antibody response. The late appearance of antibodies in the peripheral blood supports the hypothesis that not the invading L3 but rather the adult parasites elicit the production of specific antibodies. A stage-specific antibody response against the L3 was not observed. Main antigens are located in the body wall, especially in the gelatinous outer cuticle, of adult A. crassus.

Influence of flumequine and oxytetracycline on the resistance of the European eel against the parasitic swimbladder nematode Anguillicola crassus.

The effect of the antibacterial drugs flumequine (FQ) and oxytetracycline (OTC) on the defence system of the European eel (Anguilla anguilla L., 1758) was investigated using an experimentally induced infection with the parasitic swimbladder nematode Anguillicola crassus. Eight weeks after oral administration of infective larvae, the mean recovery of the parasites in FQ-treated eels was lower than in non-medicated control animals, and significantly lower than in OTC-treated eels. Mean numbers of peripheral blood granulocytes and B-lymphocytes, as well as the total number of circulating lymphoid cells, showed a significant increase as a result of the infection, while drug treatment merely affected the quantity of the lymphoid cells. The difference in protection against the parasite after FQ or OTC administration points to a modulation of the fish resistance as a result of the drug treatment. The results favour a modulation of the cellular rather than the humoral response, as no specific antibodies were found.

Influence of flumequine on in vivo mitogen responses of European eel (Anguilla anguilla L., 1758) lymphoid cells.

The influence of flumequine on mitogen induced lymphoid cell proliferation in European eels (Anguilla anguilla L., 1758) was studied. For this purpose an in vivo test, using peroral drug administration followed by successive intraperitoneal injections with concanavalin A (ConA) or bacterial lipopolysaccharides (LPS) and 5-bromo-2'-deoxyuridine, was applied. Direct counting of proliferated cells in blood smears revealed that flumequine possesses mitogenic properties. A synergistic and an antagonistic effect of the drug was observed after LPS and ConA stimulation, respectively. Flow cytometric analysis of peripheral blood lymphoid cells showed a significant reduction of the mean proportion surface immunoglobulin positive cells in the flumequine-treated animals. It is concluded that flumequine enhances proliferation of lymphoid cells (probably surface immunoglobulin negative cells) in eel under the present experimental conditions.

Production, characterisation and applicability of monoclonal antibodies to European eel (Anguilla anguilla L., 1758) immunoglobulin.

Monoclonal antibodies (mAbs) to European eel (Anguilla anguilla L., 1758) immunoglobulin (Ig) were produced, characterised and tested for applicability in a number of immuno(cyto)chemical assays. The selected mAbs, WEI 1 and WEI 2, were specifically reactive with Ig heavy and light chain, respectively. WEI 1 appeared to react with all or nearly all Ig molecules, B cells and plasma cells. WEI 2 was reactive with a subpopulation of those cells, indicating that European eel possesses at least two antigenically different light chain types. Both mAbs could be used for detection of antigen-specific antibodies in plasma by means of an enzyme-linked immunosorbent assay (ELISA).