ELISA-Based Segregation of Adult Spring Chinook Salmon for Control of Bacterial Kidney Disease: Annual Report 1991.

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (RS), a serious disease of salmonid fish worldwide. The disease has a major impact on spring chinook salmon populations in the Columbia River system. There is strong evidence that RS can be transmitted from parent to progeny, and segregation of progeny based on levels of antigen detected in adult fish may obviate this mode of transmission. Results are presented from the third year of a four year study to investigate segregation of broodstock as a tool for controlling BKD. Segregation of adult fish infected with RS has been achieved using enzyme-linked immunosorbent assays (ELlSAs) optimized in the first and second year of this project. Gametes from both 1990 and 1991 broodstock, either injected with erythromycin or receiving no antibiotic injection were successfully segregated into groups having either high or low levels of the RS soluble antigen. Offspring have been monitored every three months from the 1990 broodstock and are being monitored from the 1991 broodstock. Antigen levels in the offspring from the 1990 segregation experiment at Marion Forks Hatchery were low and clinical BKD was not observed in any of the juvenile fish. At Carson National Fish Hatchery, antigen levels were also low in fish which were sampled December 1990 through July 1991. Total mortality was low throughout these sampling periods. An increase in mortality was observed in November-December 1991, and preliminary evidence suggests that motality may have been due BKD. The epizootic appears to have equally effected both offspring from high and low RS antigen level parents. Antigen levels in moribund fish are being examined to confirm the prevalence of RS infection

Elevated temperature treatment as a novel method for decreasing p57 on the cell surface of Renibacterium salmoninarum

Renibacterium salmoninarum is a Gram-positive diplo-bacillus and the causative agent of bacterial kidney disease, a prevalent disease of salmonid fish. Virulent isolates of R. salmoninarum have a hydrophobic cell surface and express the 57-58 kDa protein (p57). Here we have investigated parameters which effect cell hydrophobicity and p57 degradation. Incubation of R, salmoninarum cells at 37 degrees C for \u3e4 h decreased cell surface hydrophobicity as measured by the salt aggregation assay, and decreased the amount of cell associated p57. Incubation of cells at lower temperatures (22, 17, 4 or -20 degrees C) for up to 16 h did not reduce hydrophobicity or the amount of cell associated p57. Both the loss of cell surface hydrophobicity and the degradation of p57 were inhibited by pre-incubation with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Cell surface hydrophobicity was specifically reconstituted by incubation with extracellular protein (ECP) concentrated from culture supernatant and was correlated with the reassociation of p57 onto the bacterial cell surface as determined by western blot and total protein stain analyses. The ability of p57 to reassociate suggests that the bacterial cell surface is not irreversibly modified by the 37 degrees C treatment and that p57 contributes to the hydrophobic nature of R. salmoninarum. In summary, we describe parameters effecting the removal of the p57 virulence factor and suggest the utility of this modification for generating a whole cell vaccine against bacterial kidney disease

Biologically Active Factors against the Monogenetic Trematode Gyrodactylus stellatus in the Serum and Mucus of Infected Juvenile English Soles

Length of survival of the monogenetic trematode Gyrodactylus stellatus in serum and mucus collected from English soles Pleuronectes vetulus at different stages of a laboratory epizootic suggests that both the mucus and serum may be involved in resistance to the parasite. In general, trematode survival was shorter in the serum and mucus samples collected from English soles at the later, recovering stages of infection. A rabbit antiserum against English sole whole serum was used in a gel diffusion immunoassay to show that mucus from infested English soles contained proteins antigenically similar to English sole serum proteins. Precipitation reactions appeared strongest in mucus collected during later stages of infection. No precipitation reactions were detected in the mucus of uninfested fish, indicating that the precipitation bands that were observed were associated with G. stellatus infections.Keywords: serum, Pleuronectes vetulus, mucus, Gyrodactylus stellatu

Development of a Vaccine for Bacterial Kidney Disease in Salmon, 1984 Annual Report.

The data presented here demonstrate that there is some variability to the antigenic structure of KDB. Although gel filtration of all antigenic preparations revealed a wide range of sizes for antigens, resolution on a denaturing gel revealed relatively few protein bands and immunological assays revealed the same (3) low number of antigens. It is of particular interest that there seems to be a protein of 60 kd in all preparations, but that there are not larger individual molecular species. This, in turn indicates that the larger molecular weight species detected in gel filtration are most likely aggregates or membrane fragments composed of a lower molecular weight subunit. Use of ultrafiltration of KDM-2 medium appears to be successful in eliminating contamination of high molecular weight material found in KDM-2. There appears to be no alteration in the number of soluble antigens produced by growth in either medium, nor in the number of proteins, as detected by SDS-PAGE. However, soluble antigens isolated from UF-KDM-2 does appear to have greater heterogeneity in their isoelectric focusing (IEF) patterns than those from UF-KDM-2. Also, although there does appear to be an extended lag period in KDB growth on UF-KDM-2, there is no alteration in final O.D. or wet weight of cells. Thus, it appears that UF-KDM-2 may be an alternate medium for those wishing to isolate purified bacterial proteins or antigens. ELISA assays have been developed for the detection of soluble KDB antigens. This system is currently being developed as a sensitive measure of the presence of soluble antigen in serum and tissues of fish. Such a sensitive assay may also allow for the detection of KD+ spawners by the testing of ovarian fluid or serum. ELISA assays have also been developed to detect antibodies to soluble and cellular antigens of KDB. These systems have been proven successful in the detection of rabbit and murine monoclonal antibodies against KDB antigens. Future work will develop the use of anti-fish immunoglobulin (Ig) reagents to detect the presence of fish antibodies to KDB. This would be an extremely useful tool to be used in monitoring the immune response of salmon to the various test vaccines. The various antigens characterized in this study, along with whole KDB cells are currently being conjugated to various immunopotentiating agents. Testing of these prototype vaccines is currently under study

Development of a Vaccine for Bacterial Kidney Disease in Salmon, 1988 Final Report.

Bacterial kidney disease of salmonids is a very complex disease which appears to exploit a variety of pathogenic mechanisms. An understanding of these mechanisms is essential to the development of efficacious vaccines. It has become well established from the studies published .in this report and those of others that soluble antigens which are secreted by Renibacterium salmoninarum have toxigenic potential. If they are found to be responsible for mortality, the development of toxoid(s) could be paramount to the production of a vaccine. One must, however, be circumspect in producing a vaccine. A thorough knowledge, not only of the pathogen, but also of the immune system of the host is an absolute requirement. This becomes of particular importance when dealing with fish diseases, since the field of fish immunology is still within its infancy. This lack of knowledge is particularly felt when the induction of a prophylactic immune response concomitantly leads to pathological side effects which may be as destructive as the original infection. Indeed, it appears that some aspects of BKD may be due to the induction of hypersensitivity reactions. If such immunopathologies are expressed, it is prudent to thoroughly evaluate the nature of the immunoprophylaxis to insure that these harmful sequelae do not occur. Evaluation of a variety of antigens, adjuvants, immune responses, and survival data leads us to recommend that attempts at prophylaxis against BKD should center upon the elicitation of cellular immunity utilizing preparations of Mycobacterium chelonii. The choice of this species of mycobacteria was made because of its effectiveness, ease of maintenance and production, and the lack of need for its propagation within containment facilities. These assets are important to consider if large scale vaccine production is to be profitable. As can be seen from the data provided, M. chelonii alone is capable of producing prophylaxis to BKD, however, this is likely due to the induction of non-specific immunity and not to the existence of crossreactive antigens. Therefore, future studies should be devoted to further work on the induction of specific immunoprophylaxis incorporating this agent

Development of a Vaccine for Bacterial Kidney Disease in Salmon, 1986 Annual Report.

Bacterial kidney disease (BRD) has been and remains a chronic contributory problem limiting the productivity of salmon of the Columbia River Basin. Control of this disease will not come easily, but it would lead to a tremendous increase in the health and numbers of salmon populations. Vaccination of salmon of Renibacterium salmoninarum (KDB) is a potentially successful method of controlling this disease. To date, however, no successful vaccine has been developed for general use. A possible solution to this problem,and thus the goal of this research, is to isolate the antigenic components of KDB and enhance their ability to activate the host defenses. This will be accomplished by the chemical modification of these antigens with potent immunomodulatory substances. These modified antigens will then be tested for their effectiveness in inducing immunity to BKD and thereby preventing the disease. The goal of the project's third year was to test the immunogenicity and prophylactic value in coho salmon (Oncorhynchus kisutch) of various chemical conjugates of Renibacterium salmoninarum cells and major antigens. This was accomplished by assessing the serum antibody response, the cellular immune response (cellular proliferation), and the kinetics of mortality after Lethal injections of the bacterium. An important facet of this research is the identification and isolation of virulence factors. These studies are not only important to the dissection of the mechanism of pathogenesis of bacterial kidney disease, but the purification of such a factor(s) will insure the production of a more potent vaccine. The studies completed this year have: (1) identified antigenic material which protect; (2) identified antigenic material which can exacerbate the disease; (3) identified a possibly major mechanism of pathogenesis via the interference with antibody; (4) the general ability to produce delineated a western blot technique for identification of infected fish; (5) described the use of monoclonal antibodies for antigenic analysis; and (6) identified an unusual and dramatic effect of R. salmoninarum cells on phagocytic function

Development of a Vaccine for Bacterial Kidney Disease in Salmon, 1985 Annual Report.

Bacterial kidney disease (BRD) has been and remains a chronic contributory problem limiting the productivity of salmon in the Columbia River Basin. Control of this disease will not come easily, but it would lead to a tremendous increase in the health and numbers of salmon populations. Vaccination of salmon to Renibacterium salmoninarum (KDB) is a potentially successful method of controlling this disease. To date, however, no successful vaccine has been developed for general use. A possible solution to this problem, and thus the goal of this research, is to isolate the antigenic components of KDB and enhance their ability to activate the host defenses. This will be accomplished by the chemical modification of these antigens with potent immunomodulatory substances. These modified antigens will then be tested for their effectiveness in inducing immunity to BKD and thereby preventing the disease. The goal of the project's second year was to chemically modify the major antigens of Renibacteirium salmoninarum, immunize coho salmon (Oncorhynchus kisutch), and to test the immunogenicity of the preparations used. Immunogenicity of the antigenic material was tested by (1) admixture experiments, using whole KD cells with muramyl dipepetide, Vibrio anguillarum extract, E. coli lipopolysaccharide, or Mycobacterium tuberculosis in Freund's complete adjuvant. In addition to these goals a number of important techniques have been developed in order to facilitate the production of the vaccine. These procedures include: (1) the use of the soluble antigen for diagnosis in the ELISA and Western blot analysis, (2) detection of salmonid anti-KD antibodies by an ELISA technique, (3) detection of cellular immune responses to the soluble antigen, and (4) development of immersion challenge procedures for bacterial kidney disease (BKD)

Polyclonal activation of salmonid B lymphocytes

The process of invitro polyclonal activation of coho salmon (Oncorhynchuskisutch) lymphocytes was examined with respect to the induction of mitogenesis, total immunoglobulin production, and the production of specific antibodies or plaque forming cells. These studies demonstrate that antigen specific stimulation of antibody production is not linked to mitogenic activity, or total immunoglobulin production, while the polyclonal activation of specific antibody production is closely linked to these functions. Stimulation of immunoglobulin production by phytohemagglutinin suggests that this mitogen may not be limited to T cell activation in salmonids or, alternatively, it may induce the production of lymphokines capable of polyclonally activating B cells. Further, fetal calf serum was found to cause production of large amounts of immunoglobulin in without antigenic stimulation

ELISA-Based Segregation of Adult Spring Chinook Salmon for Control of Bacterial Kidney Disease, Annual Report FY 1989.

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a serious disease of salmonid fish worldwide. The disease has a major impact on spring chinook salmon populations in the Columbia River system. There is strong evidence that R. safmoninarum can be transmitted from parent to progeny, and therefore culling of gametes from infected parents should obviate this mode of transmission. This report presents the results from the first year of our four year study to investigate segregation of broodstock as a tool for controlling BKD. The segregations will use Enzyme-Linked Immunosorbent Assays (ELISAs) as detection systems to identify, in tissues of infected fish, proteins produced by R. salmoninarum. A first step in the development of the described detection systems was the optimization of the production of important antigenic proteins from R. salmoninarum. Different culture media were qualitatively and quantitatively evaluated for their ability to support production of cellular and soluble proteins. The major factor affecting antigen quality was the presence and absence of calf serum. Media components and R. salmoninarum growth products could not be separated during harvest of proteins from the cultures containing serum. This caused problems with the quantitation of actual bacterial proteins in the preparation. Thus media without serum is currently employed. Two independent ELISA techniques for the identification of infected parents were examined. One technique is based on polyclonal antisera produced in rabbits and the second is based on mouse monoclonal antibodies (Mabs). To develop the latter system, several Mabs against a major R. salmoninarum antigenic protein were produced. These Mabs were used for the detection of R. salmoninarum antigens in infected fish and also to characterize proteins produced by the bacterium. Both ELISAs were deemed suitable for the segregation of parents into the high and low BKD groups required for this study. An alternate system for the detection of R. salmoninarum proteins, the Western blot, was also investigated, This technique was 100 to 1000 fold less sensitive than either ELISA system and therefore will be useful only for confirmation of highly positive tissues. Future work will attempt to increase the sensitivity of the Western blotting system. Finally, two hatcheries were identified for use in the described segregation experiments. The Carson National Fish Hatchery (Skamania County, WA) and the Marion Forks Fish Hatchery (Linn County, OR) will be used for the experiments