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In the late 1960s, Chinook salmon Oncorhynchus tshawytscha from the Green River, Washington, were successfully introduced into Lake Michigan. During spring from 1988 to 1992, large fish die-offs affecting Chinook salmon occurred in the lake. Multiple ecological factors probably contributed to the severity of the fish kills, but the only disease agent found regularly was Renibacterium salmoninarum, the causative agent of bacterial kidney disease. In this study, survival after challenge by R. salmoninarum was compared between two Chinook salmon stocks: a Lake Michigan stock from Wisconsin (WI) and the progenitor stock from the Green River. We found that the WI stock had significantly greater survival than the Green River stock. Next, the WI and Green River stocks were exposed to the marine pathogen Listonella anguillarum (formerly Vibrio anguillarum), one of the causative agents of vibriosis; survival after this challenge was significantly poorer for the WI stock than for the Green River stock. A close genetic relationship between the Green River and WI stocks was confirmed by analyzing 13 microsatellite loci. These results collectively suggest that disease susceptibility of Lake Michigan Chinook salmon has diverged from that of the source population, possibly in response to pathogen-driven selection.

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The relative efficacies of 1 commercial and 5 experimental vaccines for bacterial kidney disease (BKD) were compared through a cohabitation waterborne challenge. Groups of juvenile chinook salmon Oncorhynchus tshawytscha were vaccinated with one of the following: (1) killed Renibacterium salmoninarum ATCC 33209 (Rs 33209) cells; (2) killed Rs 33209 cells which had been heated to 37°C for 48 h, a process that destroys the p57 protein; (3) killed R. salmoninarum MT239 (Rs MT239) cells; (4) heated Rs MT239 cells; (5) a recombinant version of the p57 protein (r-p57) emulsified in Freund's incomplete adjuvant (FIA); (6) the commercial BKD vaccine Renogen; (7) phosphatebuffered saline (PBS) emulsified with an equal volume of FIA; or (8) PBS alone. Following injection, each fish was marked with a subcutaneous fluorescent latex tag denoting its treatment group and the vaccinated fish were combined into sham and disease challenge tanks. Two weeks after these fish were vaccinated, separate groups of fish were injected with either PBS or live R. salmoninarum GL64 and were placed inside coated-wire mesh cylinders (liveboxes) in the sham and disease challenge tanks, respectively. Mortalities in both tanks were recorded for 285 d. Any mortalities among the livebox fish were replaced with an appropriate cohort (infected with R. salmoninarum or healthy) fish. None of the bacterins evaluated in this study induced protective immunity against the R. salmoninarum shed from the infected livebox fish. The percentage survival within the test groups in the R. salmoninarum challenge tank ranged from 59% (heated Rs MT239 bacterin) to 81% (PBS emulsified with FIA). There were no differences in the percentage survival among the PBS-, PBS/FIA-, r-p57-and Renogen-injected groups. There also were no differences in survival among the bacterin groups, regardless of whether the bacterial cells had been heated or left untreated prior to injection.

A candidate vaccine against Aeromonas hydrophila in rainbow trout, Oncorhynchus mykiss, was developed using a bacterial lysate. To test the strength of protection, A. hydrophila challenge models were compared using injection into both the intraperitoneal (IP) cavity and the dorsal sinus (DS) with selected doses of live bacteria washed in saline or left untreated. Unlike the IP route, injection into the DS with either saline washed or unwashed cells resulted in consistent cumulative mortality and a dose response that could be used to establish a standard challenge having an LD(50) of approximately 3 x 10(7) colony forming units per fish. Survivors of the challenge suffered significantly lower mortality upon re-challenge than naïve fish, suggesting a high level of acquired resistance was elicited by infection. Passive immunization using serum from hyper-immunized fish also resulted in significantly reduced mortality indicating protection can be transferred and that some portion of resistance may be antibody mediated. Vaccination of groups of rainbow trout with A. hydrophila lysate resulted in significant protection against a high challenge dose but only when injected along with Freund's complete adjuvant. At a low challenge dose, mortality in all groups was low, but the bacterial lysate alone appeared to offer some protection.

Abstract. An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibacterium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r 2 ϭ 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r 2 ϭ 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing Ͻ3 g.Regular monitoring of the health of fish reared in intensive aquaculture is an important step toward reducing the introduction and spread of infectious diseases. 32 The immune system of fish may be adversely affected by changes in physiologic processes resulting from an inattention to water quality or fish rearing density, 4,30 diet formulation, 7,21,40 or fish handling methods. 4,5 Serum antibody levels are an indicator of the overall immune system activity of a fish. The amount of antibody can be estimated using a variety of assays, including agglutination, radial immunodiffusion, or neutralization reactions, although the sensitivity of these tests is limited. 1,27,31,41 Monoclonal antibodies to fish antibody 3,11,37 have been adapted to the enzyme-linked immunosorbent assay (ELISA) and used to quantify low levels of specific antibody activity. 2,9,24,38 When estimating fish antibody levels in an ELISA, several dilutions of each serum sample are usually analyzed to arrive at an endpoint dilution or units of antibody. 2,8,20,24,26,41 Replicate analyses of several dilutions of each test serum, however, can limit the number of fish evaluated on each ELISA microplate and increase reagent costs. In an effort to analyze greater numbers of fish on each microplate, the optical density (OD) of a single dilution of test serum has been used for antibody concentration comparisons. 13,15,18,38 because of nonrandom sources of error such as slight differences among reagent preparations or individual microplates, direct comparison of OD values is valid only for samples assayed...