Brown trout Salmo trutta injected with antigenic extracts from a pathogenic isolate of Saprolegnia parasitica developed specific antibodies that were detected by enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) and Western blotting (WB), but not by immunodiffusion (ID). Three groups of five 2 yr old brown trout were injected intraperitoneally with 3 different antigenic extracts: small hyphal fragments (HF) and soluble extracts from sonicated mycelia grown in medium with or without β-sytosterol (SEB and SE, respectively). In the 2 groups injected with SE and SEB, antibodies were found in 66.7% of the serum samples by ELISA, 54.5% by IF and 48.5% by WB. In the group injected with HF, only 1 trout survived the experiment, and in this fish only 1 sample was positive by ELISA. The results obtained by ELISA and IF were similar and show that there is cross-reaction between the antigens used. By WB, the proteins most frequently recognised were 2 proteins of 25 and 29 kDa. No significant differences were found in the groups injected with SE or SEB.
KEY WORDS: Saprolegnia parasitica · Brown trout · Antibody response · Immunological techniques
Resale or republication not permitted without written consent of the publisherDis Aquat Org 74: [107][108][109][110][111] 2007 Sohnle & Chusid 1983, Bly et al. 1993. Hodkinson & Hunter (1974) investigated the influence of culture conditions on the antigenic products of Saprolegnia and found that precipitating reactions only occurred between salmon serum and antigens from Saprolegnia grown in medium containing β-sytosterol.The aim of the present research was to study the capacity of brown trout injected with antigenic extracts of Saprolegnia parasitica pathogenic to salmonids to produce specific serum antibodies.
MATERIALS AND METHODSFish and antigenic extracts. Three different groups of five 2 yr old brown trout (225 ± 70 g weight and 26.5 ± 2.8 cm length) were injected intraperitoneally with 0.2 ml of antigenic extracts in phosphatebuffered saline (PBS) (approximately 200 µg of total protein) twice at 2 wk intervals. The extracts were mixed with Freund's complete adjuvant at a ratio of 3:1 in the first injection and with Freund's incomplete adjuvant (1:1) in the second injection. A fourth group was injected only with PBS and adjuvant as a negative control group. Three different antigenic extracts were prepared using the pathogenic isolate TRU 8 of Saprolegnia parasitica (formerly Saprolegnia sp.; Fregeneda-Grandes et al. 2000, 2003: (1) small hyphal fragments (HF) obtained after sonication of formalised-mycelial mats growing on glucose-yeast broth (GY: 1% glucose, 0.25% yeast extract); (2) soluble extract (SE) from sonicated formalised-mycelia growing on GY; and (3) SE from sonicated formalisedmycelia growing on GY supplemented with 20 mg l -1 of β-sytosterol (SEB). The experiment was carried out between April and June 2004 at a fish farm in small raceways situated in the open and with a continuous supply of well water. The water temperature varied fro...