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 Freund's 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 uptake for bath immunization and to facilitate 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.
Fish tissues and body fluids contain naturally occurring proteins or glycoproteins of non-immunoglobulin (Ig) nature that react with a diverse array of environmental antigens and may confer an undefined degree of natural immunity to fish. They consist of microbial growth inhibitory compounds that include “acute phase” proteins such as transferrins, caeruloplasmin, and metallothionein. Their action is simply to chelate metal ions and deprive bacteria and other parasites of essential inorganic ion sources. Both serum and cellular interferons are found in fish, and this anti-virus protein has been demonstrated mainly in salmonids during viral disease studies. Enzyme-inhibitors (α2-macroglobulin and other α-globulins) thus far detected in fish appear to be antibacterial proteinases, and are involved in the inhibition of extracellular proteases secreted by fish pathogens. Fish also possess a variety of relatively specific lytic molecules that cause cell lysis, and some of these materials are hydrolase enzymes (lysozyme, chitinase, chitobiase) whose main actions are against bacteria and fungi. In addition, mucus contains trypsin-like proteinases which destroy gram-negative bacteria. Nonspecific lysins and agglutinins against erythrocytes and other cellular antigens are found in serum, eggs, and skin mucus. The lysins, including toxins, some of which are bacteriolytic in activity, are, in their mode of action, natural or spontaneous, antibody-independent and noncomplement-mediated. In contrast, specific hemolytic antibodies (Ig), which complex with antigens, bind complement, and cause complement-mediated immune lysis, are reported to exist. The agglutinins are generally reactive toward certain sugar residues on erythrocyte or bacterial cell walls, and in most cases act as lectins or lectin-like molecules. Natural lysins and agglutinins behave in a similar way as antigen-induced antibodies or Igs, but exhibit a high degree of cross-reactions, due to the occurrence of similar carbohydrate determinants on many types of microbial cell surface. As with mammals, both C-type (calcium-dependent) and S-type (thiol-dependent) lectins are present in fish. They more resemble invertebrate lectins than those of higher animals. Fish lectins appear to play antibacterial or antifungal roles and in some instances seem to be involved in egg-sperm fusion, polyspermy prevention, and embryo development. Natural, non-Ig precipitins (e.g. α-precipitin and C-reactive proteins) are found largely, but not exclusively, in fish serum and precipitate with simple monosaccharides or long chain polysaccharides of certain stereochemistry and glycosidic linkages. Their functions remain unknown, but C-reactive protein is induced following stress-induction and exposure to inflammatory agents. Many of the above mentioned “defence” substances are present in skin mucus and possess the capacity to react with potentially infective microorganisms including parasites. Mucus thus acts as an immediate defence barrier to invasion and/or colonisation of pathogens.
Crustaceans have efficient means to defend themselves against most potential pathogens. Their hemocytes are crucial in these immune reactions and are capable of phagocytosis, encapsulation, nodule formation, and mediation of cytotoxicity. Recent progress in the handling of hemocytes, and the isolation and purification of several of the factors involved in the defense reactions show that the prophenoloxidase activating system (the proPO-system) and associated factors are important mediators of crustacean immunity. The use of purified factors of the proPO-system and separated hemocytes have made it possible to demonstrate in freshwater crayfish two proteins that are directly involved in cellular communication between different hemocytes. One of this proteins, a β-1,3-glucan binding protein present in the plasma, also appears to function as a recognition protein in the arthropod immune system. Another protein, a 76 kD protein derived from the granules of the hemocytes, is multifunctional and mediates hemocyte degranulation, spreading, and attachment. Also the prophenoloxidase itself and the prophenoloxidase activating enzyme, a serine proteinase, have recently been purified. Thus, it is now possible to elucidate the molecular details of the crustacean immune reactions in much more detail.
Phagocytes are cells principally dedicated to the recognition and elimination of invading organisms and damaged tissue. Those described in fish are the granulocytes (particularly neutrophils) and mononuclear phagocytes (tissue macrophages and circulating monocytes). Their movement to sites of microbial invasion is an early event in the inflammatory response and the role of host-derived factors as attractants, such as eicosanoids, is discussed. Opsonins mediate the recognition between phagocyte and particle, and receptors for serum complement component C3 and the Fc fragment of opsonic antibody have been described. Fundamental to the protection offered by the phagocytes is their bactericidal larvacidal activity, which is closely associated with the production of oxygen free radicals. Phagocytes as accessory cells are discussed, including their role in antigen presentation. A knowledge of the modulation of phagocyte function, with activation by various substances and suppression by others, is important if protective responses are to be achieved by up-regulating phagocyte activity.
Piscine macrophage aggregates (MAs), alternatively known as melano-macrophage centers (MMC), are focal accumulations of macrophages usually containing the pigments hemosiderin, lipofuscin and ceroid, and melanin. The structures are not confined to fish and have been observed in other poikilothermic vertebrates. The aggregations are most commonly present in the spleen, pronephros, mesonephros and liver but may be found in other organs, especially in relation to inflammation. In the lower fishes (Agnatha, Chondrichthyes) the pigmented cells tend to be solitary or in small (<30 cells/aggregation), irregularly shaped aggregations primarily in hepatic tissue. In the Osteichthyes, greater numbers of cells and aggregates are present than in the lower fishes. These aggregates are more nodular, and they occur more commonly in the spleen and kidney rather than the liver. An exception to these observations if found in the Clupeiformes and the Salmoniformes, considered lower teleosts, who have poorly organized and irregularly shaped aggregations that are smaller than the higher teleosts. Macrophage aggregates function in normal physiological processes and in the body's defense against injurious agents. Evidence indicates that these functions are multiple, complex, and not well understood. They may be classified as, (a) immune, including humoral and inflammatory responses; (b) storage, destruction, or detoxification of exogenous and endogenous substances; and (c) iron recycling. There is a growing body of information that elucidates these functions. Macrophage aggregates qualify as anatomical and cytological biomarkers since they are known to change in number, size, and pigment content in relation to fish health and environmental degradation. Their value lies in their ubiquity, availability, and ease of measurement. There have been few extensive, controlled attempts to produce MAs or to study their kinetics by chronic exposure to contaminants known to exist in polluted environments. Clearly, without such investigations the value of MAs as monitors (biomarkers) of fish health and environmental degradation remains questionable.
Pasteurellosis, caused by Pasteurella piscicida, is one of the most threatening diseases of wild and cultured marine fish, and has been reported from many geographical areas including the USA, Japan and the Mediterranean countries. The objective of this article is to construct a picture of the current state of knowledge about this bacterial pathogen and the pathogenesis of the disease it causes. We review some important questions such as the controversial taxonomic position of the bacterium, and its main virulence mechanisms. The epidemiology of the disease, the routes of transmission and the putative reservoirs of P. piscicida in the environment are also discussed. Finally, a detailed survey of the strategies for controlling the disease is performed, including new diagnostic procedures, chemotherapy, employment of immunostimulants, and improvements in immunization programs.
Disease resistance in fish encompasses a variety of mechanisms including maintenance of epithelial barriers and the mucus coat; nonspecific cellular factors such as phagocytosis by macrophages and neutrophils; nonspecific humoral factors such as lysozyme, complement, and transferrin; and specific humoral and cellular immunity. Numerous nutritional factors can significantly affect incidence and severity of a variety of infectious diseases. Individual micronutrients known to affect disease resistance include vitamins C, B , E, and A and the minerals iron and fluoride. Macronutrient (protein, lipid, and carbohydrate) levels have not been critically evaluated. There are indications that certain fatty acids may be important factors in disease resistance. The potential for dietary enhancement of disease resistance in fish culture certainly exists. Before this can be achieved, more information is required on pathogenesis and specific resistance mechanisms involved in individual diseases, the specific effects of various nutrients, and how these effects are modulated by other dietary components and environmental factors.
Selected features of the responses by fish to helminth parasites are discussed and comparison is made where appropriate with mammals. These include: (i) Factors influencing host specificity and consideration of the mechanisms that underpin the restriction of some parasites in their host spectrum, (ii) How fish leucocytes kill helminth larvae, with emphasis on the role of released oxygen (ROS) and nitrogen (RNS) free radicals from macrophages, (iii) Immune evasion strategies used by fish helminths, including invasion of immunologically privileged sites, encystment, adsorption of host proteins on the parasite surface, and high surface membrane turnover, (iv) Potential immunogens for vaccination and use for immonodiagnosis of infection, and (v) Natural and induced protection against helminths, with emphasis on the potential for future vaccination strategies.