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The Problem
Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum has been implicated as a significant factor in the 5-year decline of the chinook salmon populations in Lake Michigan that began during 1988. It is believed that a combination of factors, including nutritional stress due to low food availability, accompanied by high salmon densities and heavy parasite infestations, resulted in an ecosystem imbalance that likely triggered the BKD epizootics. However, the mechanisms that ultimately resulted in epizootic BKD and high fish mortality are poorly understood. A better understanding of the processes necessary for transmission of R. salmoninarum and for development of overt disease in infected fish is crucial to the success of programs to minimize the impact of the disease in Lake Michigan chinook salmon populations and assist in restoration of the important sport fishery. Objectives During 1999, researchers at the WFRC began a mult-year study in coordination with Great Lakes fishery biologists to examine the role of BKD in the continued losses of Lake Michigan chinook salmon. This project is using techniques in fish bacteriology and fish immunology to address 2 objectives: (1) To identify hatchery practices that may exacerbate the transmission of R. salmoninarum from infected to healthy fish, and (2), to investigate how the defenses of chinook salmon respond to R. salmoninarum, and if they may explain why some fish die from BKD. Four goals have been identified for the first objective of this study. They will focus on the detection and transmission of R. salmoninarum:
The second objective of this study focuses on the immune responses of chinook salmon and has 3 goals:
Methodology
To complete the first objective, the effects of various hatchery practices on the initiation and progression of R. salmoninarum infection in chinook salmon will be investigated, including weight loading and space densities used during hatchery rearing, oral administration of erythromycin to fish before release, and marking of fish with fin clips or coded wire tags before release. To remove confounding factors and provide consistent challenges of fish with R. salmoninarum, the hatchery practices will be evaluated under controlled laboratory conditions. This work requires the development of a reliable laboratory R. salmoninarum challenge that does not bypass the surface defense mechanisms of the fish (a serious drawback of injection challenges). Several variations of immersion and cohabitation challenges that have been reported will be evaluated for use in laboratory investigations of hatchery practices. Field studies will be used to validate the laboratory findings.
In conjunction with laboratory and field studies, methods for detection and quantification of R. salmoninarum in fish tissues and body fluids and in hatchery water samples will also be compared; methods used for fish samples will include serological tests that either detect the bacterium (fluorescent antibody test; FAT) or unique proteins produced by it during an infection (enzyme-linked immunosorbent assay; ELISA). The comparisons will also include a nucleic acid-based procedure, the nested polymerase chain reaction (nPCR), that detects a specific segment DNA responsible for encoding the major surface protein of the kidney disease bacterium. Fish health management programs typically rely on use of sensitive pathogen detection methods to minimize introduction and dissemination of infectious microorganisms. However, understanding the host- pathogen relationship in the context of the host may be equally important, as the fish health manager can use such information to identify aquaculture practices that favor disease resistance in a cultured fish
Highlights and Key Findings We examined the common hatchery practice of feeding juvenile salmon the antibiotic erythromycin to reduce losses from BKD. Results suggested that erythromycin feeding begun shortly after R. salmoninarum exposure (for example, during fish marking or other handling procedures) might be more effective for preventing the development of BKD than the feeding of erythromycin before a handling event. Research on BKD has been hampered by the slow growth rate of R. salmoninarum, and by the lack of an artificial challenge system that mimics one of the natural routes of infection. Previously, challenges had been done by injection of the pathogen, which usually caused death more rapidly than a natural infection, and often circumvented or overwhelmed the fishes' defenses. Our research team has designed reproducible waterborne and cohabitation challenge systems that use the sensitive ELISA and the FAT to quantify the response of fish exposed to the bacterium. Results have shown that infections can result from exposure to very low concentrations of the bacterium, although infection of >50% of exposed within a 24-hour period generally requires a higher bacterial concentration. The method has proven essential for research on the relation between stress and the horizontal transmission of R. salmoninarum, and for other work in progress concerning the pathogenesis of BKD and for testing potential control strategies. Two comparisons of various methods to detect R. salmoninarum were also completed. Kidney tissue samples selected from among the 202 male and female fish were used for these analyses. The highest prevalence was detected when the kidneys were tested by the nPCR, followed by the enzyme-linked immunosorbent assay. Five methods commonly used to diagnose R. salmoninarum infections were used in a second, and more inclusive, comparison. Precautions were taken to ensure an unbiased comparison. Samples were also coded so that actual fish number was not revealed until all testing was complete. As in the first comparison, the highest prevalences of R. salmoninarum were observed when samples were tested by the nPCR and the ELISA. New additions were made to our toolbox of assays used to monitor immune functions in fish. They emphasize aspects of the antimicrobial activities of a phagocytic cell in fish that closely resembles the mammalian macrophage. These professional phagocytes are especially important because they play a central role in recognizing and engulfing foreign pathogens, and initiating the immune response. Where Are We Headed In 2003 During FY 2003, we will continue to examine selected factors in the detection and transmission of R. salmoninarum. Our comparisons of the relative sensitivities of various methods for R. salmoninarum will continue by modifiying some existing assays so they can be used to make diagnoses without sacrificing the fish. When complete, these new sampling techniques will offer the fish health specialist a nonlethal alternative for diagnosing BKD. For the second objective of this study, we will determine the relative efficacies of different vaccines to protect chinook salmon against infection by R. salmoninarum. Groups of chinook salmon will be vaccinated with various R. salmoninarum cellular or protein-based vaccines, then challenged with the kidney disease bacterium by cohabitation with other chinook salmon previously infected with R. salmoninarum by injection. In a related study, we will use the assays in our immunological toolbox to monitor the humoral and cellular immune responses of chinook salmon following an R. salmoninarum challenge. The results of this study and the vaccine trial will provide valuable new information regarding what immune functions are most important for fish to produce a protective immune response to the bacterium. Project Contact Diane Elliott Email: diane_elliott@usgs.gov Publications |
