Douglas P. Anderson

Dietary intake of immunostimulants by rainbow trout affects non-specific immunity and protection against furunculosis.

Immunostimulant preparations Macrogard, Candida utilis, Saccharomyces cerevisiae, Evetsel, Chitosan, or FinnStim were mixed into semipurified diets and fed to groups of rainbow trout for 1 week. Fish were bled by non-lethal caudal puncture and blood samples assayed for changes in non-specific cellular immunity and humoral protein levels. In the immunostimulated fish, hematocrit levels and lymphocyte counts remained relatively stable; however, elevations were observed in oxidative radical release, myeloperoxidase activity, phagocytic indexes, and potential killing activities of phagocytic cells including neutrophils. Total plasma protein and total immunoglobulin levels were elevated by the dietary immunostimulants. A challenge with the virulent pathogen that causes furunculosis, Aeromonas salmonicida, showed that the immunostimulated groups of fish were more resistant to the disease, confirming the potential use of these substances in fish culture for the prevention of disease.

Immunostimulants, adjuvants, and vaccine carriers in fish: applications to aquaculture

Abstract Use of immunostimulants, adjuvants, and vaccine carriers in fish culture offers a wide range of attractive methods for inducing and building up protection against diseases. Immunostimulants and adjuvants can be administered before, with, or after vaccines to amplify the specific immune response generating elevations of circulating antibody titers and numbers of plaque-forming cells. Special applications of immunostimulants include assisting shower or other regimens to increase topical uptake of vaccines. In addition, immunostimulants may be used alone, inducing elevated activities in the nonspecific defense mechanisms such as increased oxidative activity of neutrophils, augmented engulfment activity of phagocytic cells, or potentiating cytotoxic cells. In cases where disease outbreaks are cyclical and can be predicted, losses may be reduced by elevating the nonspecific defense mechanisms, and the immunostimulants may be used in anticipation of events to prevent losses from diseases. Complete Freunds adjuvant was one of the first immunostimulants used in animals to elevate the specific immune response, and it has also been successfully used in conjunction with injection of fish bacterins. Other adjuvants, immunostimulants, and biological response modifiers that have been used in fisheries research include levamisole, salt baths, and bacterial lipopolysaccharides. Vaccines have been adsorbed to inert particles, such as bentonite on latex beads, to carry the immunogens to maximize in vivo uptake for bath immunization and to facilitate in vitro phagocytosis. Each substance presents special problems in timing and method of administration (injection, immersion, oral—by feed—or flush treatments), dosage adjustments for size and fish species, storage stability, and cost. An additional consideration is that the nonspecific defense mechanisms and immune responses in fish are highly variable among individuals and statistical validation requires appropriate sample numbers and carefully controlled experiments. This article reviews the literature and present concepts of use of immunostimulants, adjuvants, and vaccine carriers in fish. Cautions for use are noted, as some of these potent substances can suppress or alter biological pathways if used inappropriately. Recent research, defining pathways of the action of immunostimulants, adjuvants, and vaccine carriers, helps explain how these substances activate the protective mechanisms in fish. In addition, immunostimulants used alone hold tremendous potential for use in fish farms, hatcheries, and aquaculture facilities to reduce losses from infectious diseases. Research on the immunostimulant, levamisole, and the light oil adjuvants for use in food fish is in progress. Applications for use of these immunostimulants are proposed.

Effect of soybean protein on serological response, non-specific defense mechanisms, growth, and protein utilization in rainbow trout

Rainbow trout (Oncorhynchus mykiss) were fed chemically and immunologically defined diets containing either a low-temperature fish meal or soybean preparations containing high (SBM) or low levels of the globular antigenic proteins glycinin (G) and beta-conglycinin (BC) for 26 weeks. While trout fed both soybean preparations exhibited poorer growth than those fed fish meal, those fed the soya diet with high levels of G and BC showed the most significant growth diminution, elevated feed conversion and impaired utilization of dietary protein. Changes in intestinal morphology were also observed in fish fed the SBM diet, coinciding with the inferior nutritional performance. Several serologic and non-specific immune defense mechanisms were monitored. Cell counts showed increased leukocyte cell numbers as well as increased concentrations of plasma protein and immunoglobulin in the soya-fed fish. Increased neutrophil, monocyte and macrophage activity, as assessed by several oxidative radical production and phagocytic index assays, were higher in the soya-fed fish, possibly indicating an inflammatory or hypersensitivity response. The results of this study suggest that antigenic soya protein affects non-specific defense mechanisms, growth performance, and protein utilization in rainbow trout. Furthermore, it would appear that dietary sensitivity can be overcome by process removal of this immunologically characterized antigen in soybean protein. Serological and immunological assessments appear to be very useful criteria for evaluating various protein sources for fishes.

Prevention of stress in rainbow trout (Oncorhynchus mykiss) fed diets containing different doses of glucan

An experiment was performed to determine the effects of stress on non-specific defence mechanisms in rainbow trout fed diets containing different doses of glucan. Fish were fed with 0, 0.1, 0.5 and 1.0% glucan concentration in food. After 4 weeks of feeding, fish were stressed by 2 h transportation. The effect of stress was investigated by measuring changes in blood levels of cortisol, glucose, total protein and composition of their leukocyte population, as well as by changes in non-specific defence mechanisms of the fish. After 4 weeks feeding with glucan, elevated phagocytosis and oxidative radical production were observed in treated fish, but the levels did not correlate with the different doses of glucan. Stress induced by 2 h transportation caused high cortisol levels in plasma and hyperglycaemia in all groups, but the lowest level of glucose was measured in the group fed the low (0.1%) dose of glucan. Respiratory burst activity, phagocytosis, serum protein and lyzosyme levels were found to be significantly reduced by stress. The most dramatic reduction was observed in the control group, but the changes were not affected by glucan doses. One week post stress, hyperglycaemia was still observed in control and fish fed medium and high doses of glucan. Further reductions of total protein and intracellular oxidative radical production, were measured in all groups, but in fish fed with low dose of glucan the changes were less dramatic. The phagocytosis ratio increased in all groups, but did not attain the levels measured before stress in control and in the group fed the high concentration of glucan. A spontaneous infection with Flexibacter columnaris caused mortality in all groups except the group fed the low level of glucan. The results of the present study show that feeding of glucan in low doses several weeks before transportation can help to prevent negative effects of stress.

Glucan injection or bath exposure given alone or in combination with a bacterin enhance the non-specific defence mechanisms in rainbow trout (Oncorhynchus mykiss)

Abstract Elevations in the non-specific defence mechanisms were noted after trout were injected or bathed in glucan solutions or in solutions containing the glucan combined with a bacterin, Y. ruckeri O-antigen. Periodic non-lethal blood samples showed that neutrophil activity as evaluated by the ability of the cells to stick to glass and produce oxidative radicals as detected by the nitroblue tetrazolium (NBT) assay, rose after treatments within 2 days. Elevated phagocytosis, assessed by increased uptake of glutaraldehyde-treated sheep red blood cells, also confirmed these kinetics. The numbers of circulatory glass-adherent cells from fish given the glucans by injection or bath was twice the level of the sham-treated controls; likewise the phagocytic ratio also was two-fold higher. In following the kinetics of the non-specific defensive responses, the injection of the glucan caused an immediate, slight reduction of NBT staining cells and numbers of leukocytes before the 2–3 day rise; this was not obvious in the fish given the glucan by bath. The glucans could play an important role in the prevention of diseases in fish culture.

Neutrophil, glass-adherent, nitroblue tetrazolium assay gives early indication of immunization effectiveness in rainbow trout

Neutrophil activity in rainbow trout (Oncorhynchus mykiss) is increased upon antigenic stimulation with the Yersinia ruckeri O-antigen bacterin. The characteristics of neutrophil attachment to glass and nitroblue tetrazolium (NBT) staining were used to determine the effectiveness of immunization programs with fingerling rainbow trout. Fish immunized by intraperitoneal injection with doses of 100, 10, or 1 microgram of the bacterin showed the highest responses in that order in numbers of glass adherent, NBT-positive neutrophils. Studies on the kinetics of the occurrence of numbers of glass-adherent, NBT-positive staining cells from the fish injected with the 10 micrograms dose showed the numbers of positive cells were largest on Day 2 after injection. The specific immune response was confirmed by demonstrating the presence of plaque-forming cells by the passive hemolytic plaque assay and the rise in humoral antibody titers by passive hemagglutination 12 days after injection. The effects of immunization in trout could be detected earlier by using the neutrophil glass adherence and NBT reduction assays than by using assays based on observations of the specific immune response.

In vitro immunostimulation of rainbow trout (Oncorhynchus mykiss) spleen cells with levamisole.

An in vitro immunization and cultivation method for fish spleen organ sections was used to investigate the effects of levamisole on the immune response. After 10 days of culture with either 50 micrograms/ml, 25 micrograms/ml, 5 micrograms/ml or no levamisole in the media, the nonspecific defense reactions were measured by determining the metabolic activity of neutrophils by using the nitroblue tetrazolium test, and phagocytic and adherence indexes by incubating the fish cells with suspensions of formalin-killed Staphylococcus aureus. The specific immune system activity was shown by the passive hemolytic plaque assay demonstrating the numbers of antibody-producing cells. Elevations were found in nonspecific defense and specific immune response in the 5 micrograms/ml levels of levamisole. The 25 micrograms/ml levels instigated a slight elevation in some nonspecific levels but showed suppression in the specific immune response. The 50 micrograms/ml levels suppressed all indicators whether levamisole was given alone or combined with Yersinia ruckeri O-antigen or DNP-Ficoll. These antigens are themselves nonspecific defense stimulators, and a synergistic effect was evident when combined with 25 micrograms/ml and 5 micrograms/ml levels of levamisole.

The immune response in immunized and naturally infected rainbow trout (Salmo gairdneri) to Diplostomum spathaceum as detected by enzyme-linked immunosorbent assay (ELISA).

The enzyme-linked immunosorbent assay (ELISA) procedure was modified and adapted for detection of circulating antibodies in rainbow trout (Salmo gairdneri) against metacercariae of the digenean trematode Diplostomum spathaceum, the causative agent for diplostomiasis. Rainbow trout (Salmo gairdneri) were injected with sonicated metacercariae representing 10, 40, and 100 metacercariae per fish. Three weeks after immunization the average titers for trout injected with 10, 40, and 100 metacercariae were 874, 841, and 525, and by six weeks the titers had fallen to 299, 349, and 203, respectively. Nine weeks after initial immunization, two remaining fish initially immunized with 100 metacercariae per fish were injected with a booster of 50 sonicated metacercariae per fish. Four weeks later the average titer was 1204. Serum samples from naturally infected wild fish tested for the presence of circulating antibodies against Diplostomum spathaceum showed 25 of 27 with positive titers.

FISH IMMUNOLOGY AND FISH HEALTH

Publisher Summary This chapter reviews fish immunology and fish health. Fish hatcheries and farms are becoming increasingly important in supplementing sport fisheries and food production. However, in most facilities, the animals are kept at relatively high densities, causing well-known problems of stress and disease. From most studies, it is evident that the problems on the farms can be encountered by measures preventing disease outbreak, or by treatment of the actual disease with drugs or chemicals. As long as effective prevention cannot be achieved for all diseases, the fish farmer has no choice. In the short term, it is important to choose drugs that are not immunosuppressive. In the long term, the relationship between fish genetics and immune reactivity is an exciting new area of research. All the present data on fish-cell populations, regulation of the response by factors, and the major histocompatibility complex are needed to find out why certain fish strains differ in disease resistance.

Comparisons of nonspecific and specific immunomodulation by oxolinic acid, oxytetracycline and levamisole in salmonids

Oxolinic acid, a promising drug for the treatment of bacterial fish disease agents, was tested for possible immunomodulatory effects on fish. Another antibiotic oxytetracycline, known to be immunosuppressive at higher treatment doses, and levamisole, a known immunostimulator for higher vertebrates, were also compared for causing changes in the nonspecific defense compartment and the specific immune system in rainbow trout. Groups of fish were immunized with Yersinia ruckeri O-antigen bacterin in combination with selected doses of the drugs. The nonspecific defense activity was measured by demonstrating neutrophil metabolic activity by the nitroblue tetrazolium assay, by counting engulfed bacterial cells for a phagocytic index and by counting leukocytes with adherent bacterial cells for the adherence index. The specific immune response was monitored by the passive hemolytic plaque assay demonstrating the numbers of antibody-producing cells. The results showed that oxolinic acid, used at recommended doses for the treatment of bacterial diseases, did not cause immunosuppression in either the nonspecific defense or specific immune system compartments, whereas tetracycline at 10 mg/kg caused reduced activity in both. Fish given levamisole injections before the antigen injection showed a stimulated nonspecific defense but a much reduced specific immune response.