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The Problem Anadromous salmonids are susceptible to a variety of pathogenic microorganisms, including at least 30 bacteria and viruses. Whereas the impact of these microorganisms on salmonids in wild and natural rearing areas is difficult to measure, losses from disease among hatchery salmonids are both common and well documented. The disease state depends on 3 factors, the host (fish), the disease organism and the environment. Disease is not the result of a single event, rather the consequence of an imbalance in the host-pathogen relationship. The presence of a pathogen in a salmon population will not always result in losses due to disease and fish can remain asymptomatic carriers under favorable conditions. However, stressors exceeding the limits of a fish's physiological tolerance will diminish its ability to resist disease, and consequently, become a threat to fishes health. For the salmon, the hatchery environment is quite different than the environment they inhabit in the wild. Successful fish husbandry requires the minimization of environmental stressors such as handling, high rearing densities and poor water quality, while maintaining as high a carrying capacity as possible for the facility. Unfortunately, there is no simple measure of the health status of a fish population. Since the immune system of any higher organism is paramount for its well being, measures of the host defense provide an attractive means to quantify fish health. However, we neither understand the immune system of salmon, nor whether it modulates with seasonal and life stage changes, or differing rearing conditions.
Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD), a serious disease problem of wild and cultured salmonid fishes worldwide. Control of this bacteria by use of antibiotics is difficult due to its slow growth, and conventional vaccine strategies are ineffective and may actually worsen the disease state. Furthermore, the bacteria can be passed not only between fish in the same water system (horizontal transmission), but also from one generation to the next via infected eggs (vertical transmission). During infection, R. salmoninarum can evade immune surveillance by surviving and possibly replicating within host macrophages (a type of white blood cell). Also, R. salmoninarum produces large amounts of a unique protein (p57) which suppresses a number of immune functions. Control of R. salmoninarum will require a better understanding of the bacteria itself, the salmonid immune system and how the bacteria and host defenses interact. Objectives The immunology laboratory at the Western Fisheries Research Center is working to provide a better understanding of the salmonid immune system and how it responds to pathogenic organisms and the fishes environment. We have developed a panel assays to measure the immune functions of salmonid fishes. This panel of assays is being used to (1) provide fishery managers with measures of a salmon population's health using methods to assess the humoral and cellular immune functions of individual fish; (2) examine the effects of hatchery practices on the immune functions of salmonid fishes; (3) characterize the relationship between the salmon immune system and Renibacterium salmoninarum; and (4) study the interaction of salmon leucocytes (white blood cells) with p57, an essential virulence factor of R. salmoninarum. As new methodologies are developed for the life sciences we try to incorporate them into our panel of immunological assays. Molecular techniques for examining the control of immunologically relevant genes offer exciting possibilities for our understanding of how the salmonid immune response is controlled. We are developing molecular probes for the detection and quantitation of immunologically relevant gene products using a quantitative polymerase chain reaction (qPCR). Finally, since conventional disease control methods have not been effective for R. salmoninarum, we are working to develop efficacious control strategies and tools for salmonid fish hatchery managers to prevent outbreaks of BKD, including methods to rapidly detect infected broodstock and the development of a DNA vaccine. Methodology Assessment of the salmonid immune system is based testing various fish tissues or body fluids with a panel of serological and immunological assays. The serological assays include standard fish health measures including hematocrit, leucocrit and plasma protein concentration, as well as the total immunoglobulin concentration, lysozyme and complement activity, which are better predictors of immune system capability. Analysis of the cellular immune functions involve isolation of leucocytes from the fish, and measurements of their ability to migrate (chemotaxis), ingest foreign particles (phagocytosis), or produce bactericidal compounds (chemiluminescent response). Also, an enzyme-linked immunosorbent assay (ELISA) will be used to measure the production of antibodies to a specific protein antigen or
Studies of the interactions of the salmonid immune system with R. salmoninarum or the p57 will require culture of the bacteria and, if needed, subsequent purification of the p57. To examine the effects of BKD at the cellular level, leucocytes will be removed from the fish and cultured in the presence of the bacteria or the p57. Metabolic activities by those cultured cells in response to the stimuli will then be measured by the cellular assays. To examine changes in expression of immunologically relevant genes, molecular probes will be made to detect specific genes transcripts (messenger RNA). Quantitative polymerase chain reaction (qPCR) assays using those probes will be used to study how the fish respond to pathogenic organisms at the molecular level. Disease free stocks of fish will be obtained from a variety of sources and reared at our facility. The care and use of the animals is strictly controlled to ensure that studies involving experimental fish are not compromised by avoidable technical problems. Highlights and Key Findings We have developed a panel of immunological assays and used them for several studies which provided hatchery managers with information about the effects of various rearing practices on the defenses of salmon. A four year study was done to determine the effect of rearing temperature on the immune system of sockeye salmon throughout their life cycle. Two groups of fish were reared at 8°C and 12°C and sampled twice a year for 3 years to examine their immune functions. The results of this study indicated that salmon reared at 8°C relied more heavily on the non-specific immune response, while the specific immune response was used to a greater extent when the fish were reared at 12°C. Furthermore, as the fish matured most of the immune functions remained intact, though there were decreases in some white blood cell counts and in complement activity compared to nonmaturing adult fish. In another study we investigated how immune functions are affected by the amount of food consumed by the fish. Three groups of fish were fed daily to satiation, 70% or 40% of satiation and the immune functions were assayed when the fish had been on the ration amounts for 3 months. It was found that increased ration levels may decrease the percentage of macrophages which can ingest foreign particles but increase the white blood cell concentrations and lysozyme activity. The panel of assays was also used to study the effects of using hydrolyzed fish proteins in fish feeds as a potential means to stimulate the immune system. In this study we were unable to detect an immunostimulatory effect of the hydrolyzed protein.
The panel of immunological assays has also been used to study the host-pathogen relationship between salmon and Renibacterium salmoninarum. The effect of purified and recombinat versions of p57 on leucocytes from healthy fish was examined. Both versions of p57 suppressed several important functions of macrophages including migration (chemotaxis) and production of bactericidal reactive oxygen anions. Although the p57 protein suppresses some cellular immune functions, salmonid fishes produce a very strong specific antibody response to it. A study was completed in which a variety of protein antigens were administered to groups of rainbow trout and chinook salmon under standardized conditions. The resulting antibody responses were compared on the basis of an ELISA for antigen-specific salmonid antibody. Both fish species produced a weak antibody response to most of the antigens, which in gereral, were proteins to which the fish would never be exposed. In contrast, injection of the p57 protein elicited a strong antibody response in both species. We speculated that the relatively low diversity among salmonid antibodies is compensated by their having evolved to bind strongly to fish pathogens without extensive modification of the antibody genes as in mammals. The ability of R. salmoninarum to be passed from generation to generation via vertical, or egg-associated transmission in salmon may affect the immune functions of infected progeny fish. Exposure of a young fishes developing immune system to a foreign protein can induce a state of immune tolerance, or unresponsiveness, to that protein later in life. To examine the role of vertically-transmitted R. salmoninarum and its antigens on immune tolerance and immunosuppression, we compared chinook salmon reared from the eggs of females with either undetectable, medium or high levels of R. salmoninarum infection. Since the probability of vertical transmission is related to the maternal infection level we expected a low, medium and high infection rates in the progeny fish. Analysis of our findings suggested that transmission of R. salmoninarum only occurred among eggs from the females with very high R. salmoninarum infection levels. Fish from highly infected females had an elevated antibody response to the p57 compared to fish from the medium and uninfected females during the preinjection sampling and during some weeks after antigen injection. These results indicate that a state of immune tolerance may not have been induced in the fish reared from highly infected females. However, maternal infection may have induced some form of immunosuppression in the progeny fish. Fourteen days after challenge with Vibrio anguillarum, a bacteria which infects salmon in the ocean, total mortality among the fish from the uninfected females was 21%, compared with 45% mortality among the fish from both the medium and highly infected females. These results indicated that the progeny of even mildly R. salmoninarum infected females were more suseptable to infections by other pathogens. This study also strengthened the results of a previous study in this laboratory which recommended that hatchery operations include destruction of gametes from infected broodstock to maintain healthy stocks of salmon. Where Are We Headed In 2003 In the coming year we will be modifying our panel of immunological assays for use with striped bass, a warm-water fish species. Intensive fish culture of this species is developing, and bacterial pathogens such as Streptococcus iniae can cause significant economic losses to a hatchery. Unfortunately, little is known about the immune system of striped bass. It is therefore very important that we gain a better understanding of the host defense of striped bass so that effective control strategies can be developed for their diseases. Molecular probes are being developed for a number of genes which are relevant to the salmonid immune response. The modulation of gene expression in response to vaccination and bacterial infection will be investigated. We also hope to incorporate these molecular probes into our panel of immunological assays to gain a better understanding of how the salmon immune system functions. Project Contact John Hansen Email: jhansen@usgs.gov Publications |
