Fish immune system and its nutritional modulation for preventive health care

Animal Feed Science and Technology 173 (2012) 111–133

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Fish immune system and its nutritional modulation for preventive health care夽 Viswanath Kiron ∗ Aquatic Animal Health Unit, Faculty of Biosciences and Aquaculture, University of Nordland, 8049 Bodø, Norway

a r t i c l e

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Keywords: Fish Feeds Nutrition Health Amino acids Nucleotides Fatty acids Antioxidants Vitamins Probiotics Prebiotics Glucans Immunomodulators

a b s t r a c t Aquaculture contributes significantly to world food supplies and the rapid growth of this sector has brought forth the need to ensure that development is based on environmentally responsible practices, including those concerning feeds. The major players in the aquafeed industry are greatly aware of this and they attach importance to sustainability issues during feed development. There is consensus among the feed manufacturers and the farmers that quality feeds should not only ensure superior growth, but also return prime health. Therefore, the potential health promoting quality of each component is to be taken into account while formulating feeds. The role of dietary nutrients or additives on the functions of the immune system in fish has been investigated since the 1980s. Not all nutrients have received attention; most of the studies have been directed towards vitamins C, E and fatty acids (oils). Popular additives comprise yeast-derived products such as glucans and mannan oligosaccharides, besides probiotics. Several of these components have been examined for their ability to protect fish from stressors or diseases. The physiological outcomes attributed to these nutrients or additives are presumed to be translated to good health. More convincing evidences should be gathered before they are classified as ‘functional ingredients’. Aquafeeds of the future are expected to impart dual benefits of good growth and health to the farmed organism, and preventive health care through nutritional means is certainly a strategy to ensure sustainability in aquaculture. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The contribution of aquaculture to world food production has increased significantly over the last few decades and this sector now supplies nearly half of the total fish and shellfish used for human consumption (FAO, 2010). The prime farmed species include carps, shrimps and salmonids. Advances in culture techniques and the introduction of new species have contributed to the rapid growth of the aquaculture industry. Considering its importance in the world food sector, it is widely recognized that the industry should become sustainable from every angle. Norway, the leading aquaculture

Abbreviations: FAO, Food and Agriculture Organization; IgM, immunoglobulin M; GIT, gastrointestinal tract; GALT, gut associated lymphoid tissue; NCC, non-specific cytotoxic cells; MHC, major histocompatibility complex; IgT, immunoglobulin T; TCR, T-cell receptor; ELISA, enzyme-linked immunosorbent assay; PRRs, pathogen recognizing receptors; NO, nitric oxide; iNOS, inducible NO synthase; RNA, ribonucleic acid; DNA, deoxyribonucleic acid; RAG-1, recombination activating gene-1; IL-1␤, interleukin-1␤; EFA, essential fatty acids; LA, linoleic acid; LNA, linolenic acid; PUFA, polyunsaturated fatty acids; TNF-␣, tumor necrosis factor-␣; PG, prostaglandin; EPA, eicosapentaenoic acid; CLA, conjugated linoleic acid; HUFA, highly unsaturated fatty acids; C3-1, complement3-1; CR3, complement receptor 3; TLR, toll-like receptor; FOS, fructo-oligosaccharides; GOS/TOS, galactooligosaccharides/transgalactosylated oligosaccharides; MOS, mannanoligosaccharides. 夽 This paper is part of the special issue entitled Nutrition and Pathology of Non-Ruminants, Guest Edited by V. Ravindran. ∗ Tel.: +47 755 17399; fax: +47 755 17349. E-mail address: [email protected] 0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.12.015

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producer in continental Europe has already taken measures directed towards an eco-friendly and sustainable production for a protracted growth and development (Norwegian Ministry of Fisheries and Coastal Affairs, 2009). The downside of intensification of the farming operations has been economic losses, primarily due to infectious diseases, particularly during the early production stages. Treatment methods including approved antibiotics and chemotherapeutants are more often neither effective nor consumer/environment-friendly. Preventive measures are deemed to be sustainable and Food and Agriculture Organization (FAO) of the United Nations wants the scientific community to investigate further on: 1. ‘the role of good nutrition in improving aquatic animal health’, 2. ‘harnessing the host’s specific and non-specific defence mechanisms in controlling aquatic animal diseases’, 3. ‘use of immunostimulants and non-specific immune-enhancers to reduce susceptibility to disease’, 4. ‘use of probiotics and bioaugmentation for the improvement of aquatic environmental quality’ and 5. ‘to reduce the use of chemicals and drugs in aquaculture’ (Subasinghe, 1997). It is now widely accepted that nutritional approaches are essential to alleviate diseases among farmed aquatic animals. The concept that better nutrition leads to improved health is very familiar in humans and is applicable to aquatic animals too. Efforts have been made over the past two decades especially in the case of farmed fish species to understand the link between nutrition, immune response and resistance to diseases. Nutritional imbalances, particularly during the larval and juvenile stages have a profound effect on growth, disease resistance and survival. The link between nutrition, dietary supplements and fish health has been extensively presented in two books (Lim and Webster, 2001; Nakagawa et al., 2007). The present review covers mainly the research done during the past ten years on immunonutrition and disease resistance in fish. An overview of the fish immune system is also provided in order to appreciate how the components of the immune system can be studied as markers of the responses of fish to dietary manipulation. 2. Nutrition, feeds and health A vast amount of information on fish nutrition has been generated during the past fifty years and these have been collated in several books including that of Halver and Hardy (2002). A concrete nutritional categorization similar to that available for farmed terrestrial animals is not possible in the case of farmed fish as there are wide differences in the anatomy and physiology of their digestive systems. The current practice is therefore to use the nutritional facts from a well-studied species for other fishes. Further, the details available are not complete, particularly on the micronutrients of the major farmed species. The requirements of both macronutrients and micronutrients have generally been based on growth and deficiency symptoms (Lim and Webster, 2001; Halver and Hardy, 2002) rather than on health status indicators including immune responses and disease resistance. Moreover, the requirements are often described based on a controlled laboratory environment in contrast to the stressful and unfavourable environmental conditions in farms, which will certainly demand increased amounts of nutrients to cope with the needs of the defence mechanisms. This has been highlighted in some of the earlier reviews on nutrients and health of fish (Blazer, 1992; Lall and Oliver, 1993). It is important not only to have a sound knowledge on nutrient requirements, but also to formulate feeds optimally using the appropriate ingredients. This has been a challenge for the aquatic feed industry, mainly because the prime ingredients, fishmeal and fish oil, have always been a constraint due to their high demand and pricing, not to mention the increased awareness on their unsustainability. Besides selection of appropriate raw materials, correct formulation and processing ensure that feeds attain physical and chemical properties suitable for the farmed animal. Any imbalance in formulation or the inferior nature of an ingredient may inadvertently impair the health status of the fish and increase their susceptibility to diseases. Proper feeding practices also have a key role in keeping the culture environment clean and reducing the chances of disease outbreaks. The concept of maintaining the health of fish through the best possible nutrition is well-accepted in modern fish farming. Scientific evidence gathered over the past thirty years indicates that dietary nutrients as well as additives could stimulate the immune system of fish and help to fend off diseases. Even so, the supporting information is scanty compared to the knowledge on terrestrial animals. Quality feeds are branded not only in terms of their nutritional features, but also based on their health promoting and disease preventing properties. As aquafeeds evolve, more of the ‘functional feeds’ would be out in the market and farmers could face the issue of being unable to make the right choice of a feed. Therefore it will be very important to verify and quantify the bioavailability and functionality of the potentially active ingredients that are incorporated, in order to present the true value of feeds. 3. Overview of the fish immune system In this review the term fish refers to the abundant and diverse bony fish (teleosts), mainly those that are farmed. The immune system of species such as rainbow trout, Oncorhynchus mykiss and common carp, Cyprinus carpio has long since been investigated. This knowledge has diversified greatly due to the variety of fish that are farmed and the inclusion of species such as zebrafish to the animal model repertoire in comparative immunology studies (Yoder et al., 2002; van der Sar et al., 2004). From an evolutionary point of view, fish are considered as the earliest class of vertebrates having both innate and adaptive immunity, though the latter defence mechanism is not as elaborate as in higher vertebrates (Warr, 1995). The fish immune system operates at the crossroads between innate and adaptive responses and is habituated to the environment and the poikilothermic nature of the fish (Tort et al., 2003). Fish are found in diverse and even extreme aquatic environments

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and they operate their defence mechanisms efficiently (Plouffe et al., 2005). They are in constant interaction with their surroundings and therefore could easily encounter potential pathogens. In the wild, fish are able to protect themselves with the help of a complex innate defence mechanism that may be constitutive (already present) or responsive (inductive) (Ellis, 2001). However, in the farms the infection pressure would be much greater due to the commonly adopted intensive culture practices. For instance, primary defence barriers such as the mucus and epidermis that constitute the local immune mechanism could be compromised through physical abrasions, providing easy access for the pathogens to the tissues. Systemic innate responses can take over at this stage, nonetheless pathogens are adept at evading these responses and infecting a fish that is generally weak. Thus the operation of the immune system is both at the local and systemic level. Despite certain differences, fish depend on both cellular and humoral immune responses as in higher vertebrates and have organs dedicated to immune defence. With the exception of lymphatic nodules and bone marrow which are functionally replaced by the head kidney, most of the generative and secondary lymphoid organs seen in mammals are also found in fish (Press and Evensen, 1999). These organs are briefly described here. The head kidney (pronephros) is aglomerular and bifurcates at the anterior part and penetrates beneath the gills (Zapata et al., 1996). This organ participates in phagocytosis and antigen processing; besides helping the formation of immunoglobulin M (IgM) and immune memory through melanomacrophage centers (Herraez and Zapata, 1986; Dannevig et al., 1994; Brattgjerd and Evensen, 1996). Further, the head kidney serves as an endocrine organ releasing corticosteroids and other hormones and is central to immune-endocrine interactions. Spleen, generally plays a secondary role compared to the head kidney in the specific and non-specific defence mechanisms. This organ is mainly made up of blood cells, endothelial cells, reticular cells, macrophages and melanomacrophages. In addition to its involvement in hematopoiesis, it is engaged in the clearance of macromolecules, antigen degradation/processing and the production of antibody. Located at the dorsolateral region of the gill chamber, close to the opercular cavity and diffused among the muscle tissues is the thymus, another immunologically important organ that produces T lymphocytes and antibody generating B cells (Zapata and Amemiya, 2000). Melano-macrophage centers or macrophage aggregates that are presumed to be primitive analogues of the germinal centers of lymph nodes are primarily seen in the stroma of hematopoietic tissue of spleen and kidney, and also in the liver of certain fish. They contain lymphocytes and macrophages as well as a variety of pigments. These aggregates trap antigens and present them to lymphocytes, sequester cellular degradation products, melanins, free radicals and catabolically broken down products (Agius and Roberts, 2003). Liver does not have a significant role in the immune defence system; nevertheless it has been shown to produce some of the acute phase reactants (Demers and Bayne, 1994). As part of the reticuloendothelial system, they scavenge the substances eliminated from circulation, though the efficiency varies between fish species (Dalmo et al., 1997). Skin, gills and gut are the mucosal tissues associated with the immune system of fish. Skin is considered as the primary barrier that provides the physical and chemical protection in association with its mucus. The latter comprising of glycoproteins, proteoglycans and proteins, constitutes a layer that exists as an interface between the fish and the environment (Dalmo et al., 1997). Diverse antimicrobial factors found in the mucus inhibit the colonization of the integument by potentially harmful microorganisms (Alexander and Ingram, 1992; Ruangsri et al., 2010). Gills are multifunctional – primarily a respiratory organ, they are also involved in the immune defence through the mucosa associated lymphoid tissues that harbour macrophages, neutrophils, lymphocytes and mast cells/eosinophilic granulocytes (Pratap and Wendelaar Bonga, 1993; Reite and Evensen, 2006). Lymphoid cell aggregations (T cells) similar to those described in mammalian mucosa are found at the interbranchial septum at the base of gill filaments, suggesting their involvement in immune surveillance of gill infections (Haugarvoll et al., 2008). The gastrointestinal tract (GIT) is an organ with multiple functions in nutrition as well as immunity. The organisation of the gut associated lymphoid system of teleost intestine is not as complex as that in mammals, but has a more diffused pattern (Rombout et al., 2011). The gut mucosa is rich in immune cells such as lymphocytes, plasma cells, eosinophilic (mast cell-like) granulocytes, and macrophages and can elicit local responses (Press and Evensen, 1999). The GIT mucosal surface is a natural interface where the intestinal microbiota and antigen cross-talk with the host fish (Montalto et al., 2009). The microbes, either commensal or pathogenic, are in direct contact with the gut mucosa and the gut associated lymphoid tissue (GALT) distinguishes between them to initiate either tolerance or immune response. The cells supporting the fish immune system are constituted of circulating white blood cells which share functional and morphological similarities with mammalian lymphocytes, granulocytes and monocytes (Zelikoff, 1998). The immune responses initiated upon injury or upon pathogenic invasion will entail phagocytosis and inflammatory processes (Corbel, 1975), ably assisted by non-specific immune cells such as monocytes/macrophages, neutrophils and non-specific cytotoxic cells (NCCs). It should be noted that phagocytic activity has been reported for trout B lymphocytes that are specific immune cells (Li et al., 2006). Eosinophilic granular cells (granulocytes) present in the mucosal region of gut and gills are capable of responding to bacteria and parasites (Secombes, 1996). Non-specific cytotoxic cells are present in the blood as well as in the lymphoid tissues and mucosal sites, and they respond to virus-infected host cells and protozoan parasites (Secombes, 1996). The humoral components of the non-specific response inhibit adherence and colonization of microorganisms and occur in serum, mucus, skin, gills and intestine. They include various antimicrobial agents such as trypsin, lysozyme, antibodies, complement factors and other lytic factors (Alexander and Ingram, 1992). Lysozyme is one of the defence factors widely occurring at body surfaces exposed to the environment including gills and intestinal tract, besides being found in circulation in blood. It is considered as a mucolytic enzyme of leucocytic origin (Saurabh and Sahoo, 2008), but is known to be synthesized in liver as well as in extra-hepatic tissues (Bayne and Gerwick, 2001). Among the leucocytes, neutrophils and monocytes and to a certain extent macrophages are associated with lysozyme (Saurabh and Sahoo, 2008). Comparing different species,

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Lie et al. (1989) pointed out that kidneys have the highest level of lysozyme, followed in the descending order by alimentary tract, spleen, skin mucus, serum, gills, liver and muscle. This enzyme has anti-inflammatory and antiviral properties, besides its high potential for bactericidal or bacteriolytic activity against Gram-positive and Gram-negative bacteria (Saurabh and Sahoo, 2008). Considering its association with leucocytes this enzyme is a preferred marker of the immune response. Complement system has a primary role in the innate immunity of fish, facilitating chemotaxis, opsonisation and pathogen destruction (Holland and Lambris, 2002), but is also linked to the acquired immune system since complement activation enhances B cell proliferation (Morgan et al., 2005). Among the several complement components (C3, C7, C4, C5 and factor B) found in teleosts, C3 is essential for all complement pathways. It is a vital constituent of blood and lymphatic and extravascular fluids in different organs and tissues (Løvoll et al., 2007). They are primarily synthesized in the liver, but extra-hepatic tissues such as head kidney, skeletal muscle, gills, skin, intestine, spleen and pylorus also produce different complement components at lower, but biologically significant amounts (Løvoll et al., 2007). The specific (acquired) immune defence mechanisms of fish include both cell- and humoral-mediated responses as in mammalian systems and the specific set of responses could be a negative memory or tolerance as well as a positive anamnestic memory (Ellis, 1977). Fish are endowed with immunoglobulins, major histocompatibility complex (MHC), T-cell receptors, and T- and B-lymphocyte populations which can elicit specific immune responses against a diversity of antigens. The lymphocyte populations of teleost fish are analogous to the mammalian T cells and B cells in many ways (Clem et al., 1991). B lymphocytes and plasma cells, principally located in spleen and kidney of fish are capable of producing antibodies and as in mammals they bear the immunoglobulin on their cell membrane (Zelikoff, 1998). Until lately it was believed that fish produced only a single class of Ig which closely resembles mammalian IgM, and that there was no distinction between teleost mucosal and systemic Ig. In 2005, genomic analysis performed on rainbow trout and zebrafish led to the discovery of a new immunoglobulin isotype, IgT (Danilova et al., 2005; Hansen et al., 2005). Recently, it has been revealed in rainbow trout that IgT can serve as a mucosal intestinal immunoglobulin and is expressed on the surface of a newly identified B cell subset that occurs in the gut associated lymphoid tissue (Zhang et al., 2010). T-lymphocytes carry a different type of antigen specific receptor, the T-cell receptor (TCR). The presence of specific cytotoxic T cells in teleosts was established based on graft rejection and cell-mediated cytotoxicity studies (Nakanishi et al., 2002). More evidence gathered during the recent years employing genomic tools have led to the identification of several key T cell markers (CD4, CD8, CD3, CD28, CTLA-4) and cytokines (interleukins, interferons and macrophage-activating factors, tumor necrosis factor, transforming growth factor, chemotactic factor and macrophage migration inhibition factor), indicating the existence of discrete regulatory T subtypes (Manning and Nakanishi, 1996; Castro et al., 2011). Thus the teleostean cell-mediated immunity is analogous to that in higher vertebrates, considering both the variety of cell types and the activity of the effector cells. For a better understanding on the specific defence mechanisms in fish, the following reviews could be useful: (Kaattari and Piganelli, 1996; Manning and Nakanishi, 1996; Nakanishi et al., 2002; Kaattari et al., 2009; Castro et al., 2011). 4. Assessing immune responses and disease resistance from a nutritional context Our understanding of the immune system of fish is incomplete with respect to the interactions between cells and organs as well as with other systems like the endocrine system. It involves various cell types and humoral factors that orchestrate a spectrum of mechanisms both at the systemic and local level. In order to document the status and functionality of the immune system, a stream-lined approach measuring an array of parameters that are indicative of the animal’s response is needed. Though in vivo responses are better representatives, ex vivo approaches based on isolated cells are often employed. Carefully designed challenge experiments are a means to study the immune defences as well as to understand the whole animal response, in terms of its stress endurance or its capacity to resist diseases. Besides genetic and environmental factors, the nutritional status of the fish can be considered as a major aspect that influences the immune responses, modulating the resistance to infection. In well-nourished humans, qualitative changes in macronutrients as well as supplementation of single nutrients influence the immune functions (Albers et al., 2005). Comparing the existing information on fishes, we understand that immunonutrition studies have been largely based on single nutrients, employing selected humoral and cellular immune markers and disease challenge models. Ideally, the approach should cover the range from a whole fish response to those at the tissue or cellular level and at the mechanistic level. A pathogen exposure study with mortality/morbidity as end point or a stress challenge with post-exposure stress mapping is indicative of the integrated host-defences. Several studies including those from our group (rainbow trout: Kiron et al., 1995; Atlantic cod: Caipang et al., 2008, 2009) have relied on this approach to test the efficacy of different dietary components in fish. However, the caveat is that these experiments need to be carefully planned and skillfully executed. Details such as the lineage of the fish and the pathogen strain employed or the type and duration of the stressor applied could influence the outcome of such studies. The reliability of these techniques is very much dependent on their repeatability. Another in vivo approach would be to measure the specific antibody production in response to vaccination, after subjecting the fish to different dietary treatments. Though this is an integrated in vivo measure indicative of both immune function and vaccine efficacy, it is not frequently employed in fish nutrition studies. When adopting this technique, one should take note of the vaccination history of the fish, besides being aware that there would be high and low responders to vaccination within a group of fish (Wiegertjes et al., 1996; Schrøder et al., 2009). The antibody response of fish is generally determined as antibody titers employing techniques like agglutination, precipitation or ELISA. However, the usefulness of this indicator in fish, compared to other vertebrates, may be questionable due to the generation of specific redox forms of IgM that is

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necessary to react with a pathogen (Kaattari et al., 1998). Therefore, a high antibody titer may not be an effective indicator if the particular redox form is not serologically distinguishable from other forms (Tort et al., 2003). Next in line are the ex vivo assessments, conducted subsequent to a prescribed feed regime, to study the innate and adaptive responses using in vitro assays. Though the in vitro component is devoid of the multiple factors that govern an in vivo measurement, the ex vivo studies would reveal differences reflecting feed effects to a reasonable extent. These observations may generate mechanistic information to test a research hypothesis (Albers et al., 2005). The ex vivo studies are mainly performed with primary cells harvested from the immune organs such as head kidney, spleen or intestine of fish subjected to different dietary treatments. Assessing the functions of the phagocytes is a reliable measure of the animal’s ability to combat bacterial infections. The added advantage is that these assays would cover not only phagocytosis per se, but also measure oxidative burst and killing activity. Further, depending on the availability of tools, one could also specifically examine the different phagocytic cell types such as neutrophils and monocytes. Natural killer cell functions have rarely been used as a dietary marker in fish studies (Inoue et al., 1998), but in mammalian literature they are considered as relatively sensitive to diet and stress and are representative of non-MHC related cytotoxic function (Albers et al., 2005). In addition to the aforementioned techniques, inflammatory responses can be studied based on the cells collected from blood, or immunerelated organs, and if the cell types are known, one could even comprehend cell-specific cytokine profiles and mediators of inflammation. As genomic resources become more available, at least in the case of important fish species, one may be able to get a better view point on the role of pathogen recognizing receptors (PRRs, i.e. toll-like receptors) in relation to antigen presenting cells. Blood or serum based assays constitute yet another battery of measurements that are relied on to evaluate primarily the innate responses. The commonly examined humoral (circulating) components are complement activity, lysozyme activity, total Ig, acute phase proteins, cytokines, while the cellular components are leucocytes and lymphocytes. These measures are also indicative of the animal’s dynamic in vivo responses. Lymphocyte proliferation is a representative assay for acquired immune cell functions and is employed to determine the overall responsiveness of T-cells. Very few studies have actually examined the dietary effects on lymphocyte proliferation in fish (Verlhac and Gabaudan, 1994; Kiron et al., 2011). Lysozyme is considered as an important index of innate immunity since it has antiviral, antibacterial and antiinflammatory properties (Saurabh and Sahoo, 2008). It is influenced by several external factors and therefore has been frequently employed in fish nutrition research. The complement system, on the other hand, is a complex enzyme cascade constituted by several glycoproteins that normally exist in their proenzyme forms. This system has two distinct pathways – classical and alternative – and signals to the host the presence of potential pathogens and helps in clearing them, besides coordinating the development of an acquired immune response. Unlike in mammals, the alternative complement pathway titers in teleosts are quite high and have the capacity to mediate the lysis of target erythrocytes from several species, suggesting their greater capacity to recognize a wider range of foreign surfaces (Boshra et al., 2006). These features, along with their potential to function at varying temperatures suggest that complement is a powerful defence mechanism in fish. Their activity was found to be influenced by external factors including nutrition in fish (Thompson et al., 1995; Kiron et al., 2004; Panigrahi et al., 2005).

5. Immunonutrition Fish health is dependent on what fish eat or better it depends on what they are fed with in the case of aquaculture. An appropriate feed and feeding regime gives optimum health; this conjecture is based on our comprehension of the linkage between the nutrition and immunology. Strictly speaking one cannot define health just associating the two aforementioned disciplines; an integrative approach to understand mechanisms would include other areas such as biochemistry, physiology, microbiology and pathology. Though fish nutrition and fish immunology has existed as separate areas since 1960s, the scientific community developed a binary thinking only during the late 1980s. The last decade has witnessed a spurt in research in this area, aided not only through cross-disciplinary efforts and the availability of modern genomic tools, but also due to the greater understanding of preventive health and the central role of feeds in keeping fish healthy. In order to generate information that is valuable for the feed industry, we need to appreciate the significance of what is currently known and try to bridge the knowledge gaps. The teleost immune system is well-developed to operate an efficient defence procedure against unfavourable situations in farms that could be either a stress factor or a pathogen invasion (Fig. 1). Under such circumstances, biological processes are activated in the fish to create a hostile milieu for the pathogen or to resolve the imbalances resulting from a stressor. The protective mechanisms such as the regulatory cytokines, the antioxidant defences, acute phase proteins or the cellular responses are initiated to tackle the situation and later terminated upon achieving their objectives. The endogenous sources of nutrients supply the basic requirements for the immune system to realize its functions as well as to protect tissues from collateral damage. Immunonutrition is aimed to provide the animal with additional resources/molecules that would support one or more of the processes broadly outlined above, to finally obtain a higher degree of protection. The existing knowledge on immune responses of fish to a nutrient, a feed ingredient or an additive is skewed towards those based on a few selected substances. The information reviewed here covers mainly the literature that appeared during the past ten years.

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Fig. 1. The concept of immunonutrition in preventive health.

5.1. Nutrients Nutrients, essential or non-essential, either singly or in combination, directly or indirectly can influence immune functions and fish health. Our understanding on the role of dietary nutrients on fish health is largely based on nutrients such as vitamins C and E and lipids. It is surprising that energy-macronutrient intake, an aspect of great importance in human nutrition, has not been addressed in fish. It may partly be due to the fact that commercial aquafeeds generally contain essential nutrients in excess of the dietary needs of the animal. Rather than problems related to macronutrient deficiencies, the high energy of the present-day aquafeeds and ingredients employed may inadvertently cause micronutrient imbalances that could compromise the functioning of the immune system. Nutritional deficiencies are seldom reported from farms; however undetected subclinical deficiencies may possibly have a link to losses due to diseases that often occur during production cycles. This aspect has been presented in a review by Hardy (2001). 5.1.1. Proteins, amino acids Dietary protein provides animals’ need for essential amino acids, nitrogen required for the synthesis of non-essential amino acids as well as nitrogen-containing physiologically relevant molecules (Young, 2000). Insufficient intake of proteins or amino acids ultimately affects the cells’ protein content and eventually incapacitates them. Ten essential amino acids are required by fish, approximately in proportions found in their bodies (Hardy, 2001). Feeds based on good quality fishmeal satisfy the amino acid needs of fish. However as replacement levels of fishmeal in feeds rise, the content and bioavailability of amino acids from alternative sources may cause concern. The inclusion of plant proteins in feeds of carnivorous fish, particularly from a source that does not appear in the food chain, could lead to amino acid imbalances. Further the endogenous antinutritional factors present in feedstuffs from plants, which if not eliminated through processing and biotechnological methods, may be toxic to fish (Tacon, 1995). These issues may cause immune dysfunctions even though such effects of antinutritional factors need convincing evidence. In gilthead seabream, Sparus aurata the inclusion of plant proteins at over 75% replacement caused liver steatosis, accompanied by a decrease in complement activity (Sitjà-Bobadilla et al., 2005). Although, there was no change in lysozyme activity linked to the plant protein inclusion, an increase in leucocyte intracellular killing ability was evident as indicated by the oxidative radical production and higher myeloperoxidase content in plasma. The authors attributed the latter observation to the inflammatory conditions in the intestine caused by the plant proteins as they noted the infiltration of granular eosinophilic granulocytes in the gut submucosa. In an earlier study on rainbow trout, a reduction in the activity of macrophages was linked to increasing levels of soybean protein in feed (Burrells et al., 1999). Soybean induced enteritis in Atlantic salmon has been investigated by different groups. Rombout and co-workers noted the transformation and migration of eosinophilic granulocytes that bear lysozyme in their granules during dietary soyabeaninduced inflammatory process (Urán et al., 2009). Krogdahl and her team examined the link between soybean induced enteritis and the susceptibility of Atlantic salmon, Salmo salar to infection (Krogdahl et al., 2000). Fish that received soy molasses had increased levels of lysozyme and IgM in the mid and distal intestinal mucosa, elevated inflammatory response and greater susceptibility to furunculosis. In contrast, a fishmeal replacement study on the same species (dehulled lupin meal was used to substitute 40% of the fishmeal protein) did not reveal any changes in the various humoral immune parameters (lysozyme and antiprotease activity, neutrophil oxygen radical production and plasma immunoglobulin) and differences in the resistance to Vibrio anguillarum in a challenge trial (Bransden et al., 2001). On the other hand, when soybean meal was replaced with cotton seed meal in feeds for channel catfish, Ictalurus punctatus, improved resistance against Edwardsiella

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ictaluri infection was observed, along with increased macrophage chemotaxis and agglutinating antibody titers (Barros et al., 2002). Amino acids have a central role in the defence mechanisms since they are involved in the synthesis of an array of proteins such as antibodies and in the control of key immune regulatory pathways. In human and animal nutrition, arginine, glutamine and cysteine have received greater attention, though several amino acids are involved in sustaining immunocompetence and disease resistance. Amino acid imbalances as well as their antagonisms could affect nutrient utilization and can have a direct consequence on immune organs and responses (Li et al., 2007). These include the activation of T and B lymphocytes, natural killer cells and macrophages; cellular redox state, gene expression and production of antibodies, cytokines and cytotoxic substances (Li et al., 2007). In mammals nitric oxide (NO) synthesis by inducible NO synthase (iNOS) in macrophages and neutrophils is an important immune mechanism against a wide range of pathogens (Bronte and Zanovello, 2005). Modifications in the expression and activity of arginase, resulting from the competition between arginase and iNOS for a common substrate directly affects NO generation by leucocytes and in turn the killing of bacteria (Kepka-Lenhart et al., 2000; Gobert et al., 2001). The role of dietary amino acids on fish immune response has hardly received attention with the exception of arginine. In a study on channel catfish, though the dietary arginine levels did not distinctly correlate with NO generation in macrophages, it was pointed out that the plasma arginine may partially regulate the intracellular availability of arginine and sequentially affect the ability of NO production by macrophages (Buentello and Gatlin, 1999). A related study disclosed that plasma amino acid concentrations were indicative of the usage of arginine and glutamine when encountering pathogens and arginine upto 2 g/kg in feeds resulted in improved disease resistance (Buentello and Gatlin III, 2001). Dietary nucleotides are considered non-essential due to the high rates of their de novo synthesis (e.g. RNA and DNA) that takes place in the human body, compared to the actual intake (Grimble and Westwood, 2000). Cosgrove (1998) has described nucleotides as “ubiquitous intracellular compounds involved in the vital cell function and metabolism – as nucleic acids, in biosynthetic pathways, in transferring chemical energy, as co-enzyme components and as biological regulators”. The relationship between dietary nucleotides and immune functions has been reviewed by Gil (2002) – they influence lymphocyte activation and proliferation, enhance phagocytic activity, influence immunoglobulin responses during early life and augment production of cytokines, particularly in the intestine. In their review on nucleotide nutrition in fish, Li and Gatlin III (2006) have dealt with, inter alia, their influence on innate and adaptive immunity in fish and have pointed out that the dietary nucleotides would support lymphoid tissues that have limited de novo synthesizing capacity. The benefits of dietary nucleotides have been demonstrated in both marine and freshwater fish. The reported improvements upon nucleotide feeding were increased complement and lysozyme activity, phagocytosis, and superoxide anion production in carp, (Sakai et al., 2001) and higher oxidative radical production by neutrophils in hybrid striped bass, Morone chrysops × M. saxatilis (Li et al., 2004). Nucleotides were found to exert an immunopotentiating effect on antibody production in tilapia (Ramadan et al., 1994) Atlantic salmon (Burrells et al., 2001) and hybrid striped bass (Li et al., 2004). In a study on turbot, Scophthalmus maximus dietary nucleotide supplementation enhanced the gene expressions of IgM and recombination activating gene-1 (RAG-1) in gill and spleen and that of interleukin-1␤ (IL-1␤) in kidney (Low et al., 2003). Studies on different fish species have revealed that dietary nucleotide supplementation enhanced their resistance to parasites, bacteria and virus. Atlantic salmon receiving dietary nucleotides were infested with fewer sea lice and were more resistant to infectious salmon anaemia virus (Burrells et al., 2001). Rainbow trout also demonstrated better survival upon challenge with infectious pancreatic necrosis (IPN) virus (Leonardi et al., 2003). Resistance towards bacterial pathogens has been reported in common carp (Sakai et al., 2001) and hybrid striped bass (Li et al., 2004). However in a recent study on early juveniles of Atlantic cod (Gadus morhua), nucleotide supplements were not found to be supportive for stress responses, though it did improve growth (Lanes et al., 2010). 5.1.2. Lipids, fatty acids Lipids are the energy dense macronutrients in feeds that fulfil both energy and the essential fatty acid (EFA) requirements. Investigations conducted during the early part of the twentieth century revealed that dietary fat is essential for the health of warm-blooded animals (Ziboh, 2000); similar observations on cold-blooded animals appeared thereafter (Castell et al., 1972; Takeuchi and Watanabe, 1977). Like other vertebrates, fish should obtain the precursor fatty acids (linoleic acid, LA, 18:2n-6 in the case n-6 family and linolenic acid, LNA, 18:3n-3 in the case n-3 family) or their highly unsaturated metabolic derivatives from dietary sources. It is known that in mammals, several polyunsaturated or monounsaturated fatty acids are involved in different immune functions, exerting their influence through changes in membrane fluidity, eicosanoid synthesis, formation of lipid peroxides, regulation of gene expression, apoptosis, alteration of antigen presentation, or modulation of intestinal microbiota (Puertollano et al., 2008). These mechanisms in turn have significant roles in inflammatory processes and the resistance of the host against infectious microorganisms. The impact of dietary lipids on the immune functions and pathogenesis in fish has been reviewed by Lall et al. (2002). In a recent review, Trichet (2010) highlighted the significance of fatty acids in building the immunocompetence of farmed fish. The increased use of alternative oil sources, particularly plant oils, responding to the challenges resulting from declining marine (fish) resources has resulted in the dilution of natural EFA sources in fish feeds. The physiological mechanisms of fish, particularly in the carnivorous varieties, have been challenged by the presence of n-6 and 18-carbon polyunsaturated fatty acids (PUFA) from the plant sources. Several studies have examined the effects of various oils and the general opinion is that different immune functions will be compromised when fish oil is replaced by plant oils in diets (Kiron et al., 1995, 2011; Bell et al., 1996; Montero et al., 2008), though there are also reports on the contrary (Gjøen et al., 2004; Seierstad

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et al., 2009). Besides the aforementioned studies that have looked at the direct impact, particularly on the cells of immune system, attempts have also been made to observe the effect on the lipid mediators – eicosanoids. In a study on Atlantic salmon, dietary safflower oil brought about three to seven fold increase in n-6 fatty acid derived eicosanoids, but it did not influence the phagocytic activity of head kidney macrophages or stress and disease resistance when compared to fish receiving fish oil (Gjøen et al., 2004). Another study on Atlantic salmon that considered the replacement of fish oil with rape seed oil revealed that there were no differences in expression of pro-inflammatory cytokines (tumor necrosis factor-␣ (TNF-␣) and IL-1␤) and in the respiratory burst responses, despite a five-fold reduction in n-3/n-6 fatty acid ratio of leucocyte membranes (Seierstad et al., 2009). A feeding study employing different plant oils, rich in either n-3 PUFA (linseed oil) or n-6 PUFA (safflower oil) compared the responses of rainbow trout to a bacterial antigen (Kiron et al., 2011). With the exception of reactive oxygen production that was significantly higher in the linseed oil fed fish, the humoral (alternate complement activity and lysozyme activity) and cellular (phagocytic activity and lymphocyte proliferation) immune responses were not affected by the oil offered. The authors concluded that the two oil types were capable of sustaining the immune responses of this freshwater fish. In gilthead seabream, substitution of fish oil by plant oils rich in oleic acid, LA and LNA led to higher amounts of prostaglandin E2 (PGE2) derived from arachidonic acid, but to lower amounts of eicosapentaenoic acid (EPA) derived PGE3 (Ganga et al., 2005). The same report inferred that in marine fish, EPA has an important role as the precursor of prostaglandin, and its deprivation could lead to alterations in the functions of leucocytes. In European seabass upon partial replacement of dietary fish oil the number of circulating leucocytes and the macrophage respiratory burst activity were significantly affected, while there were no significant differences in prostaglandin production (PGE2 and PGF2␣) (Mourente et al., 2005). Though most of the studies on lipids and fish health have considered the impact of alternate oil sources, there is a dearth of information on the effects of individual fatty acids. Improved immune responses were noted in rainbow trout that were offered purified fatty acids in diets (Kiron et al., 1995); fish that received n-3 PUFA had enhanced phagocytic activity, antibody production and better resistance to virus infection. The benefits of dietary conjugated linoleic acid (CLA) on seabass included an enhancement of bacterial defences based on alternate complement pathway analysis and increased lysozyme activity (Makol et al., 2009). It should be mentioned that feeding studies employing individual fatty acids are rare due to the costs of such formulations. Studies conducted during the 90s (reviewed by Balfry and Higgs, 2001) have shown the ability of dietary n-3 and n-6 highly unsaturated fatty acids (HUFAs) to improve disease resistance. High levels of n-3 HUFAs in diet were reported to decrease immune competence and disease resistance (Erdal et al., 1991; Li et al., 1994), but other reports (Sheldon and Blazer, 1991; Ashton et al., 1994; Thompson et al., 1996) suggested the opposite. A balance between dietary n-3 and n-6 HUFA may create the most favourable immune responses (Fracalossi and Lovell, 1994) and resistance to diseases (Kiron et al., unpublished). Feeding soy oil based diets (50 and 100% replacement) to Atlantic salmon did neither affect transport stress nor their susceptibility to infection by Aeromonas salmonicida (Gjøen et al., 2004). In another study on the same species, feeding with fish oil, rather than with canola oil, resulted in better survival upon challenge with V. anguillarum (Carter et al., 2003). In Atlantic salmon, replacement of fish oil with sunflower oil at different levels revealed significant differences in cumulative mortality upon challenge with V. anguillarum, but the observations on the negative effects of fish oil replacement were inconclusive (Bransden et al., 2003). An earlier report on channel catfish suggested that dietary lipids (menhaden and linseed oils) influenced mortality upon exposure to E. ictaluri at higher temperatures (28 ◦ C vs. 17 ◦ C) and related this finding to competitive inhibition of arachidonic acid metabolism by n-3 fatty acids (Fracalossi and Lovell, 1994). It should be stressed that no one specific nutrient is solely responsible for influencing the immune system. For instance the membranes of the immune cells while being patterned in accordance with the dietary fatty acid provisions will need several micronutrients such as antioxidant vitamins and carotenoids to act in concert to prevent oxidative damage and maintain their integrity (Chew, 1996). This aspect has been investigated in rainbow trout using dietary vitamin E (Puangkaew et al., 2004, 2005). In both the studies, the inclusion of vitamin E at 100 mg/kg diet was adequate with respect to immune and antioxidant defence mechanisms. A study on Nile tilapia, Oreochromis niloticus, compared the response of fish to differing lipid and vitamin levels in the diet (Lim et al., 2009). Significant improvement in lysozyme activity was recorded with 200 mg vitamin E/kg diet while complement activity was enhanced even at 100 mg vitamin E/kg; the latter was also influenced by the dietary lipids. Both lipids and vitamins did not have an effect on the resistance of Nile tilapia to Streptococcus iniae challenge as well as on the antibody titer against the bacterium.

5.1.3. Antioxidant micronutrients Antioxidant micronutrients comprise vitamins A, C and E, carotenoids and minerals such as selenium, zinc, copper, manganese and iron. Vitamin and mineral premixes included in aquatic feeds generally meet the requirements of the fish, notwithstanding the fact that the animal could also obtain them from the ingredients per se. Their deficiencies commonly reported in aquaculture could be due to the antagonistic interaction with other dietary ingredients that lower their bioavailability (Hardy, 2001). Erroneous dietary inclusion and unsuitable manufacturing and storage conditions can also lead to deficiencies. The stability of microingredients such as vitamins and carotenoids are impacted upon by the extrusion process during feed manufacture (Riaz, 2000; Anderson and Sunderland, 2002). Nutritional deficiencies reported from farms are

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often linked to vitamins C and E (Hardy, 2001), and therefore it is not surprising that these two nutrients are frequently studied in relation to their effects on the immune response.

5.1.3.1. Vitamin C. Vitamin C or ascorbic acid is one of the powerful reducing agents available to cells, serving as a cofactor for the incorporation of molecular oxygen into various substrates (Englard and Seifter, 1986). Several attempts have been made to study the effects of ascorbic acid on the immune responses of fish, particularly salmonids and catfish (Dabrowski, 2000). From an immunological perspective it is interesting to note that vitamin C concentrations are high in head kidney, spleen and leucocytes of fish (Gabaudan and Verlhac, 2001). Therefore it is likely that the cells of the immune system rely on this vitamin for achieving protection from pathogenic invasion. The improved oxidative burst response of leucocytes from rainbow trout was correlated to the intracellular levels of vitamin C arising from its high dietary intake (Verlhac et al., 1995). Another interesting aspect of vitamin C is its role in collagen synthesis – by functioning as a coenzyme in the formation of hydroxyproline they help to maintain the integrity of fish skin and deny entry to pathogens (Gabaudan and Verlhac, 2001). This aspect however needs further attention. The deprivation of vitamin C can lead to multiple effects including leukopenia and gill hypertrophy as demonstrated in adult catfish, Clarias gariepinus (Adham et al., 2000). Both non specific and specific immune mechanisms have been evaluated in fish in relation to dietary supplementation of vitamin C. The effectiveness of high-doses of this vitamin in augmenting immunocompetence has been evaluated by several authors (reviewed by Verlhac and Gabaudan, 1997). A vast majority of them demonstrated an enhancing effect on phagocytic activity, oxidative burst, lysozyme activity and alternative pathway complement activity. Some of the most recent reports on the non-specific responses are on bagrid catfish, Mystus gulio (Anbarasu and Chandran, 2001); yellow croaker, Pseudosciaena crocea (Ai et al., 2006); Japanese seabass Lateolabrax japonicus (Ai et al., 2004); golden shiner, Notemigonus crysoleucas (Chen et al., 2004); grouper, Epinephelus malabaricus (Lin and Shiau, 2004, 2005); milkfish, Chanos chanos (Azad et al., 2007) and rohu Labeo rohita (Misra et al., 2007). The specific cellular defence was also affected by dietary vitamin C: it influenced lymphocyte proliferation in rainbow trout (Gabaudan and Verlhac, 2001) and exhibited greater response in delayed hypersensitivity test in catfish (Anbarasu and Chandran, 2001). Different trends have been reported for antibody production in relation to vitamin C. When rainbow trout were stressed after feeding low and high amounts (44 mg vs 3170 mg vitamin C/kg diet), the fish receiving the lower level had higher antibody production (Thompson et al., 1993). In milkfish, the antibody production was augmented when the diets were supplemented with 1500 mg of vitamin C/kg diet (Azad et al., 2007). The potential of vitamin C to help fish fight diseases has been presented in a review (Lim et al., 2001c). It is obvious that the deficiency of vitamin C leads to immune suppression and susceptibility to infectious diseases. On the other hand, numerous studies support that vitamin C at levels above their normal growth requirement enhances immune response and offers resistance against diseases. The difficulty in drawing common conclusions on the protective effects of dietary vitamin C has also been discussed by Gabaudan and Verlhac (2001). Some of the information on the improved disease resistance of fish upon fortification of diets with vitamin C is presented here. Two separate studies have reported better survival of Indian major carps: (i) in the case of mrigal, Cirrhinus mrigala the survival upon challenge with Aeromonas hydrophila was nearly 80% for fish receiving high amounts of vitamin C (1014 mg/kg feed) (Sobhana et al., 2002); (ii) in the case of rohu challenged with E. tarda, survival was 70% for fish fed 500 mg of vitamin C/kg feed (Sahoo and Mukherjee, 2003). An effect of the duration of feeding vitamin C (500 mg/kg feed) was revealed in the case of Nile tilapia, O. niloticus upon challenge with A. hydrophila. Relative percent survival was 58% after 1 month and 72% after 2 months of feeding vitamin C (Ibrahem et al., 2010). In grouper and croaker a range of vitamin C levels up to 500 mg/kg feed was tested; grouper that received 288 mg of the vitamin recorded 100% survival when challenged with V. carchariae (Lin and Shiau, 2005), while only 54% of croaker that received 500 mg vitamin C survived the challenge with V. harveyi (Ai et al., 2006). The amount of vitamin reported for the different studies may be higher than what was actually present in the diet at the time of feeding as the content depends on the stability of the vitamin. Aquaculture operations such as transport, crowding and handling could be stressful and immune-depressive. The possibility of alleviating these adverse effects through feeding vitamin C has been investigated. The secretion of stress hormones by interrenal and chromaffin cells is accompanied by a high utilization of ascorbic acid and hence greater dietary supplies of the vitamin during stressful periods would facilitate efficient functioning of the immune and endocrine systems (Gabaudan and Verlhac, 2001). The capacity of dietary vitamin C to build tolerance to intermittent hypoxic stress was demonstrated in Japanese parrotfish, Oplegnathus fasciatus (Ishibashi et al., 1992). High doses of the vitamin C helped to keep the mortality low among fish subjected to the stressor. Another study examined if dietary vitamin C could influence stress and immune responses of rainbow trout exposed to temperature fluctuations (Kiron et al., 2005). Though increasing levels of the vitamin positively influenced complement activity and lymphocyte proliferation, these improvements were not sustained when the fish were subjected to temperature stress. Additional evidences are needed to establish the role of vitamin C in supporting fish under stress.

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5.1.3.2. Vitamin E. Vitamin E compounds (tocopherols) are the major chain-breaking antioxidants. They help maintain interand intracellular homeostasis of labile metabolites such as oxidizable vitamins and unsaturated fatty acids (Halver, 2002). It is known that inadequate amounts of dietary vitamin E would result in non-specific cell degenerative conditions. The greater fragility of erythrocytes is an indicator of inadequate levels of this physiological antioxidant (Kiron et al., 1994). The free radical quenching ability of vitamin E enables it to protect the PUFA of membrane phospholipids from the singlet oxygen generated during the oxidation process. As a therapeutic nutrient, high dietary vitamin E has been found to increase lymphocyte counts, stimulate cytotoxic activity of cells, phagocytosis and mitogen responsiveness in mammals (Watson, 1998). Further, it is presumed to be involved in disease prevention by hindering infection-induced increases in tissue prostaglandin production and by regulating cytokine production (Watson, 1998). Vitamin E and its immunological roles in fish have received frequent attention ever since Blazer and Wolke (1984) reported the negative effects of its deprivation on non-specific and specific immune responses of rainbow trout. Early research on this vitamin has been reviewed by Blazer (1992). Approaches taken to examine the effect of vitamin E are similar to those described for vitamin C and some of these studies have also considered the combined effect of vitamin C and different lipids/fatty acids. ˜ and co-workers. Dietary The effect of vitamin E on gilthead seabream has been investigated in different studies by Ortuno intake of 1200 mg vitamin E/kg feed (compared to 600 mg level) for 30–45 days enhanced serum haemolytic activity and the phagocytosis of head kidney leucocytes, although leucocyte migration and respiratory burst activity remained unaffected ˜ et al., 2000). A combination of vitamin E and vitamin C (at 1200 mg and 3000 mg/kg diet respectively) not only (Ortuno enhanced complement activity and phagocytic activity but also improved the respiratory burst activity; the latter parameter ˜ et al., 2001). The observations made when different stress factors was not affected when vitamin E alone was offered (Ortuno were applied on fish that received vitamins C and E, either alone or in combination did not distinctly support the hypothesis ˜ et al., 2003). that the short-term dietary administration of high doses of these vitamins could reduce stress in the fish (Ortuno The effect of chronic and acute stress was studied in the same species by comparing fish that received vitamin E (150 mg/kg diet) and those that did not. Deprivation of vitamin E resulted in higher cortisol levels and a depletion in complement activity. Under chronic stress there were no distinct changes in immune responses examined, while under acute stress the production of oxygen radicals was diminished in fish that was on the vitamin E deficient diet (Montero et al., 2001). The effect of vitamin E supplementation (0, 100 mg and 450 mg of vitamin/kg diet) on the kinetics of macrophage recruitment and giant cell formation in pacu, Piaractus mesopotamicus, maintained at different stocking densities (5 and 20 kg/m3 ) was reported by Belo et al. (2005). Fish that received the highest amount of vitamin had significantly improved cellular responses. The impact of vitamin E deprivation was evident at the highest stocking density, as these fish had fewer inflammatory cells. The phagocytic functions of gut leucocytes and head kidney macrophages were evaluated in rainbow trout after they were offered diets with different levels of vitamin E (0, 28 mg and 295 mg/kg diet) for 80 days (Clerton et al., 2001). Enhancement of phagocytosis was recorded for fish on high dose of vitamin E; the effect being significant for gut leucocytes. The oxidative burst activity of the cells from the two organs did not exhibit any relationship to the dietary vitamin levels. They concluded that the effect of vitamin E could be larger for the local responses in the intestine than for the systemic responses in the head kidney. The effect of vitamin E in relation to PUFA types on the immune responses was examined in rainbow trout (Kiron et al., 2004). Two levels of vitamin E (100 mg and 1000 mg/kg feed) were compared by supplementing them in diets with oils that differed in their n-3 and n-6 PUFA profiles. Based on the enzyme assays and measures of antioxidant status, it was suggested that the higher dose of vitamin E may have exerted a pro-oxidant effect causing higher superoxide anion production from head kidney leucocytes and greater lysozyme activity. In another related study on rainbow trout from the same research group, lower vitamin E levels were noted in head kidney when dietary n-3HUFA levels were high and this finding emphasized the importance of dietary vitamin E in supporting the immune organs under oxidative stress (Puangkaew et al., 2004). The aforementioned study also pointed out that 100 mg vitamin E/kg feed, a level slightly above that required for normal growth, was adequate for supporting humoral and cellular immune responses. Thus, the rationale for including vitamins at amounts over their normal requirement levels should consider the potential benefits, their interaction with other nutrients and the condition of fish. Supporting the hypothesis on the interdependence among nutrients, it was shown that under crowded conditions, deprivation of vitamin E led to higher mortality, particularly when the diet also contained HUFA (Trenzado et al., 2007). 5.1.3.3. Carotenoids. The functions of carotenoids at the subcellular level have been well-studied in humans, particularly linking to mitochondria. They protect the cells against oxidative injury and ensure optimal cellular functions including apoptosis, cell signaling and gene regulation (Chew and Park, 2004). The immunoprotective functions of the carotenoids depend very much on the equilibrium between the intra- and extracellular milieu and on the type and concentration of the carotenoid. Despite the role carotenoids have in the nutrition of several fishes and crustaceans (Bjerkeng, 2008), only few studies have considered them in relation to the health of the organism. It has been reported that the stimulatory potential of dietary astaxanthin and vitamin A was low in rainbow trout though their absence negatively impacted the responses studied (Thompson et al., 1995). An ensuing report on the larvae of Japanese parrotfish (O. fasciatus) and spotted parrotfish (O. punctatus) that received ␤-carotene-enriched rotifers indicated better proliferation of lymphocytes and improved survival (Tachibana et al., 1997). In rainbow trout, activities of lysozyme, complement and phagocytes and non-specific cytotoxicity were elevated upon ␤-carotene and astaxanthin supplementation (Amar et al., 2001). The study observed an enhanced

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effect when the diets contained vitamins A, C and E too. In a subsequent investigation, the same researchers validated the benefits of carotenoids derived from marine algae Dunaliella salina and red yeast Phaffia rhodozyma (Amar et al., 2004), as they improved humoral as well as cellular responses. 5.1.3.4. Minerals. Even though mineral needs of fish are generally met from the rearing environment, mineral deficiencies have been one of the earliest studied aspects in fish nutrition (Gaylord and Marsh, 1914). In higher vertebrates, minerals such as calcium, phosphorus and magnesium (linked to hard tissue mineralization), iron (linked to hemoglobin), selenium (linked to glutathione peroxidase), copper, zinc, and manganese (linked to antioxidant enzymes) have been examined in connection with their immunological roles. The review by Lall and Lewis-McCrea (2007) describes the roles of important vitamins and minerals in relation to skeletal metabolism and pathology in fish. It should be noted that in fish not much effort has been made to look at specific minerals in relation to their immunological roles. Iron has been studied for the reason that there exists a delicate balance between the need for this mineral in host defences and the requirement by microorganisms for their growth. Imbalances would compromise the immune system and the resistance of fish to diseases (Lim et al., 2001a). Zinc, another trace element directly affects the production and function of leucocytes and indirectly affects the immune system by acting as a cofactor of enzymes that influence the organ functions in man (Rink, 2000). Research conducted on dietary zinc and immune response and disease resistance in fish has been reviewed by Lim et al. (2001b). Observations reported include enhanced chemotaxis of macrophages, a lower phagocytic ability, improved or attenuated disease resistance and reduced or negligible effect on antibody production. Though not via dietary sources, zinc has been shown to be immunomodulatory both for cellular and humoral parameters (Sanchez-Dardon et al., 1999) including inflammatory responses (transcriptome profiling) and complement C3-1 protein expression (Hogstrand et al., 2002). Selenium is another important trace element for fish because it is a constituent of selenoproteins and has structural and enzymatic roles similar to glutathione peroxidase (the antioxidant enzyme). This mineral modulates the immune functions such as inflammation and virulence development (Rayman, 2000). In channel catfish selenoyeast and selenomethionine were better selenium sources compared to sodium selenite, and increase in antibody titer corresponded to their dietary concentrations (Wang et al., 1997). Improved macrophage chemotactic response and protection against E. ictaluri were also achieved in channel catfish that obtained 0.4 mg selenium/kg from the organic sources. Very little work has been undertaken in fish with this mineral and these have been included in a review by Lim et al. (2001b). The involvement of phosphorus on the immune mechanisms in fish is not clear, but it is suspected that it may supply the energy required for antibody production and phagocytic activity. Hypophosphataemia could lead to low leucocytic ATP levels and affect the disease resistance capacity of fish (Sugiura et al., 2004). Inclusion of phosphorus at 0.4 or 0.85 mg/kg in the feeds of channel catfish resulted in no mortality upon challenge with E. ictaluri (Eya and Lovell, 1998). 5.2. Immunonutrition – additives Additives are intentional inclusions to the feeds, acknowledging their capability to modulate the immune system, alleviate stress or ward off pathogens. These physiologically active compounds or biological response modifiers have been aptly termed by the European Food Safety Authority as zootechnical additives that ‘favour the performance of animals by providing good health’ (European Food Safety Authority, 2008). Additives could be either immunopotentiators (positive influence) or immunosuppressors (negative influence; but not necessarily ‘negative’ as in the case of regulatory functions) that attenuate the consequences of pathogenic invasion and contribute to welfare of the recipient fish. These substances could include intact microbes (e.g. probiotic organisms), microbial cell components (e.g. lipopolysaccharide, muramyldipeptide), fungal polysaccharide (e.g. zymosan, ␤-glucan), phytotherapeutic agents used in human and veterinary medicines (e.g. rosemary, astragalus root) and synthetic compounds (e.g. levamisole). On the other hand, micronutrients can also be considered as additives if they are supplemented in feeds at levels higher than the animals’ normal requirement. Apart from nutrients, additives intended to produce food fish of a desired quality are in vogue in the aquafeed industry. Collectively all these substances are now referred to as functional ingredients and feeds containing them are termed functional feeds. This concept is gaining foot-hold in the industry and as farmers embrace sustainable ways of farming, functional feeds serving specific needs of the growing animal are becoming more popular. In a review published over a decade ago, immunostimulants employed in aquaculture were categorized based on their sources (Sakai, 1999). They may have the potential to directly trigger the innate defence mechanisms of fish through their action on cell receptors and/or on genes linked to the immune system (Bricknell and Dalmo, 2005). The aforementioned authors suggested that the definition of immunostimulant should not be restricted to aspects related to mononuclear phagocyte system alone, but rather encompass the pattern recognition receptors of different leucocytes. Their proposed definition is – “an immunostimulant is a naturally occurring compound that modulates the immune system by increasing the host’s resistance against diseases that in most circumstances are caused by pathogens”. The rationale is that receptors on the target immune cells recognize the immune-stimulatory substances as high-risk molecules and induce defence pathways. These immune-potentiating substances are generally included as dietary supplements during stressful aquaculture operations, such as grading, transfer, vaccination or during crucial life stages to help the animal to ward off pathogens and maintain good health. The present review will only cover additives that are of substantial interest for the aquaculture industry, rather than the entire spectrum of immunostimulatory compounds examined in fish. The information is presented in three

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sections. The first one deals with the non-digestible polysaccharides – glucans and alginates that are predominantly employed as immune-modulators in fish. The second section on prebiotics covers mainly non-digestible carbohydrates that primarily promote the beneficial population of gut bacteria. The third section on probiotics highlights their application as novel therapeutics to fortify the defence system of fish. The reader is directed to earlier reviews of Sakai (1999), Bricknell and Dalmo (2005) and Trichet (2010). 5.2.1. Immunomodulatory polysaccharides Glucans include diverse glucose polymers that differ in the position of glycosidic bonds, which can be short or long, branched or unbranched, ␣ or ␤ isomers and soluble or particulate (Goodridge et al., 2009). In particular they refer either to a linear one with (1 → 3)␤-d glycosidic linkages or a branched one with side chains bound by (1 → 6)␤-d glycosidic linkages. Immunomodulatory glucans commonly employed in studies [(1 → 3)␤-d-glucan] originate from yeast (e.g. Saccharomyces cerevisiae), bacteria (curdlan from Alcaligenes faecalis), seaweed (laminarin from Laminaria digitata) and mushroom (lentinan from Lentinula edodes) (Goodridge et al., 2009). They have either pro- or anti-inflammatory effects on immune cells depending on the type of the cells and receptors involved (Goodridge et al., 2009). ␤-glucans act on different immune receptors including Dectin-1, complement receptor (CR3) and toll-like receptor (TLR-2/6), triggering macrophages, neutrophils, monocytes, natural killer cells and dendritic cells (Chan et al., 2009). A review (Dalmo and Bøgwald, 2008) has focused on how ␤-glucan modulates the immune system, besides describing their aquaculture applications both as dietary supplement and as vaccine adjuvants. The information on dietary administration of ␤-glucan to fish has been summarized in Table 1. Some of the recent work is presented here. The administration of a commercial glucan preparation significantly enhanced macrophage superoxide anion production in pink snapper (Pagrus auratus), during winter (Cook et al., 2003). The authors suggested that this approach could provide increased protection during seasons when fish are more prone to infections. In seabass (Dicentrarchus labrax), serum complement titer and lysozyme activity were significantly enhanced upon short term administration of ␤-glucan (1 g/kg feed for 15 days), and the stimulatory effect lasted for up to 30 days (Bagni et al., 2005). A combination of ␤-glucan (10 g/kg feed) and LPS (2.5 g/kg feed) offered as 3 doses during a two-week period to common carp (C. carpio) enhanced oxidative burst and bacterial killing capacity of leucocytes, besides providing protection upon challenge with A. hydrophila (Selvaraj et al., 2006). They also recorded higher antibody titers following vaccination with A. hydrophila antigen. When different levels of dietary ␤-glucan were tested on fingerlings of rohu, L. rohita (Misra et al., 2006) lysozyme and complement activity and bactericidal activity of serum rose to the highest levels on day 42 in fish that received 250 mg ␤-glucan/kg diet. This level of glucan also provided protection for the fish when challenged with A. hydrophila and E. tarda. These reports reiterate the immunostimulatory roles of ␤-glucan, particularly at the cellular level, and its capability to protect both warm and cold water fish species from diseases. The prophylactic potential of these compounds has lead to the application of yeast-based glucans in aquafeeds. Nevertheless, we need to have additional evidences on how the defence mechanisms are modulated by the glucans. Alginates are found as capsular polysaccharides in soil bacteria or as constituents of cell walls of brown algae (Phaeophyceae). They are a family of linear unbranched polysaccharides with (1 → 4) linked ␤-d-mannuronic acid and ␣-l-guluronic acid residues in different ratios (Gombotz and Wee, 1998). Usually they are extracted from seaweeds (Macrocystis pyrifera, Ascophyllum nodosum or different types of Laminaria), though bacteria of genera Pseudomonas and Azotobacter too produce these substances. The application of alginates in feeds, for larval rearing or for on-growing, benefits the fish by stimulating the innate immune system. Different types of alginates have been found to improve disease resistance in fish. Alginate derived from the seaweed Ascophyllum nodosum elevated the levels of lysozyme in Atlantic salmon indicating its immunostimulatory potential (Gabrielsen and Austreng, 1998). A commercial product (alginates from extracts of Laminaria digitata and Ascophyllum nodosum) has been evaluated in different studies mentioned here. In a long term experiment on European seabass (D. labrax), a pulse feeding of the alginates for 15 days was followed by a withdrawal period of 45 days (Bagni et al., 2005). The innate immune parameters varied in their response periods – serum complement activity was elevated by 15 day post-feeding (pf), the lysozyme activity and gill and liver heat shock protein concentrations were elevated only at 30 day pf; all these changes did not exist anymore at 45 day pf. Over the long-term period, no significant differences were observed for both innate and specific immune parameters. The effects of the same alginate product have also been examined in two separate studies on rainbow trout. Immune enhancement based on the expression of cytokines (IL-1␤, IL-8) and toll-like receptor 3 (TLR3) in spleen (Gioacchini et al., 2010) and the stimulation of cytokine genes in liver (IL-1␤, IL-8 and TNF-␣2) was indicative of a better response to a vaccine (Gioacchini et al., 2008). Others have studied the effect of sodium alginates on the immunity in groupers. When offered at 2 g/kg feed to fingerling orange-spotted grouper, Epinephelus coioides, it improved the non-specific immune indices and survival upon challenge with Streptococcus sp. and iridovirus (Yeh et al., 2008). In the case of brown-marbled grouper, E. fuscoguttatus, both humoral and cellular components of innate immunity were enhanced at a dose of 10 g sodium alginate/kg feed, accompanied by better resistance to V. alginolyticus (Cheng et al., 2008). The form of the alginates has also been reported to have an effect on their efficacy. An early report on common carp revealed that alginates from Macrocystis pyrifera produced better survival which was ascribed to mannuronic/guluronic ratios of the alginates (Fujiki et al., 1994). High M-alginate, having a high content of mannunoric acid, has been tried as an immunostimulant for the larvae of different marine species with varying success. While a significant resistance to vibriosis was recorded for Atlantic halibut Hippoglossus hippoglossus larvae fed high M-alginate bioencapsulated in Artemia (Skjermo and Bergh, 2004), the results from first feeding and weaning of Atlantic cod (Gadus morhua) larvae (Skjermo et al., 2006) and wolffish, Anarhichas minor fry (Vollstad et al., 2006) were not encouraging. Though alginates have a certain degree of

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Table 1 Parameter-wise summary of the immune responses of different fishes upon dietary administration of ␤-glucan. Fish

Response with respect to control fisha Enhanced

Unaltered

Lysozyme activity Rohu1 Large yellow croaker2 Rainbow trout3,7 Nile tilapia4 Seabass5 Channel catfish6 Complement activity Rohu1 Large yellow croaker2 Seabass5 Channel catfish6 Rainbow trout7 Carp8 Phagocytic activity Rohu1,9 Large yellow croaker2 Asian catfish10 Respiratory burst Rohu1 Large yellow croaker2 Rainbow trout3,7 Carp8 Asian catfish10 Bactericidal capability Rohu1,9 Antibody response Rainbow trout3 Nile tilapia4 Rainbow trout7 Rohu11 Asian catfish12 Pathogen resistanceb Large yellow croaker2 Rainbow trout3 Channel catfish6 Carp8 Rohu11 Asian catfish12

Vibrio harveyi Infectious hematopoietic necrosis virus Edwardsiella ictaluri Aeromonas hydrophila Edwardsiella tarda Aeromonas hydrophila

Adapted from Dalmo and Bøgwald (2008). 1 Labeo rohita (Misra et al., 2006); 2 Pseudosciaena crocea (Ai et al., 2007); 3 Oncorhynchus mykiss (Sealey et al., 2008); 4 Oreochromis niloticus (Whittington et al., 2005); 5 Dicentrarchus labrax (Bagni et al., 2005); 6 Ictalurus punctatus (Welker et al., 2007); 7 O. mykiss (Verlhac et al., 1998); 8 Cyprinus carpio (Selvaraj et al., 2006); 9 L. rohita (Sahoo and Mukherjee, 2001); 10 Clarias batrachus (Kumari and Sahoo, 2006a); 11 L. rohita (Sahoo and Mukherjee, 2002); 12 C. batrachus (Kumari and Sahoo, 2006b). a Grey shading suggests a response. b The pathogens employed are indicated in the respective boxes.

acceptance by the industry, there is limited information to support its use as an effective functional ingredient. Further in-depth studies, unravelling the mechanisms, similar to those attempted in the case of ␤-glucans would be necessary to firmly establish the benefits for aquaculture species. 5.2.2. Prebiotics In human and animal nutrition, application of prebiotics is an alternate strategy to selectively manipulate the desirable intestinal microbiota by offering non-digestible food ingredients. Roberfroid (2007) defined prebiotic as “a selectively fermented ingredient that allows specific changes, both in composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health”. Several of the feed components including starch, dietary fibers, proteins and lipids may have prebiotic properties though currently oligosaccharides are strictly classified as prebiotics. Potential prebiotic oligosaccharides are categorized based on their chemical components and the degree of polymerization. They include manno-, pectic-, soybean-, isomalto-, xylo-oligosaccharides, lactulose, inulin, fructo-oligosaccharides (FOS) and galacto-oligosaccharides/transgalactosylated oligosaccharides (GOS /TOS). Majority of the human studies have focused on the last three types (Macfarlane et al., 2008). These prebiotic substances imitate the eukaryotic cell surface receptors of

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the host to trick the virulent bacteria to adhere to them (Macfarlane et al., 2008; Shoaf et al., 2006). Prebiotics have also been studied in combination with probiotics (synbiotics) as they stimulate the growth of the probiotic and render them capable of competing with the indigenous species. There is increasing evidence in animals including fish that prebiotics can also induce immune responses. Realizing the potential benefits of prebiotics in fish, different research groups started to study their possible applications. A great deal of information is found in two reviews (Merrifield et al., 2010; Ringø et al., 2010). This section shall briefly cover some of the studies related to fish. The most common prebiotic evaluated in fish are the mannanoligosaccharides (MOS) which are glucomannoproteincomplexes derived from the cell wall of a yeast (S. cerevisiae) (Sohn et al., 2000; Merrifield et al., 2010). Though there is no general agreement on beneficial immune responses, distinct changes in the gut microbiota on MOS supplementation as well as changes in intestinal morphology such as increased intestinal absorptive surfaces, microvilli length and increase in acid mucin secreting cells have been recorded (Dimitroglou et al., 2009; Torrecillas et al., 2011). Another prebiotic preparation based on partially autolyzed brewer’s yeast, dairy ingredient components, and dried fermentation products has received much attention. Dietary inclusion of this prebiotic improved innate immune responses such as neutrophil oxidative radical anion production and intracellular superoxide anion production in head kidney macrophages from hybrid striped bass (Li and Gatlin III, 2004, 2005). However, the alternative complement activity in golden shiners Notemigonus crysoleucas was not affected by the prebiotic preparation (Lochmann et al., 2009). In the study of Li and Gatlin III (2004, 2005), mentioned above, reduced mortality upon challenge with bacterial pathogens – Streptococcus iniae and Mycobacterium marinum was noted. However, the dairy-yeast prebiotic, did not enhance resistance of goldfish, Carassius auratus, to the bacterial pathogen and did not greatly alter microbiota of the anterior or posterior gastrointestinal tract (Savolainen and Gatlin, 2009). A similar observation on the microbial community was also reported for red drum (Burr et al., 2009). Through prebiotic feeding, alteration of unculturable intestinal bacteria and exclusion of potential pathogenic bacteria were achieved in hybrid striped bass (Burr et al., 2010). Inulin [a heterogeneous blend of fructose polymers with mostly ␤(2 → 1) linkages (Niness, 1999)], is generally derived from plants. Studies on the use of inulin in fish are not as extensive as those for MOS. Inulin at a relatively high dose (150 g/kg) in the feeds of Arctic charr, Salvelinus alpinus resulted in pathological conditions in the distal intestine (Olsen et al., 2001), reduced intestinal bacterial population and composition, and even did not help preferential colonization by inulin-utilizing bacteria (Ringø et al., 2006). A study on red drum, Sciaenops ocellatus revealed that inulin (10 g/kg) feeding resulted in a single dominant organism in the intestinal microbial community (Burr et al., 2009), concordant with an earlier study on Atlantic salmon where the microbial diversity was reduced (Bakke-McKellep et al., 2007). The suitability of inulin may be further questioned since a significant inhibition of phagocytosis and respiratory burst occurred when gilthead seabream was offered inulin at 5–10 g/kg feed (Cerezuela et al., 2008). Levan, another polymer of fructose, which has mostly ␤(2 → 6) linkages (Han, 1990) is derived from plants, yeasts, fungi and bacteria. This soluble dietary prebiotic substance is being considered as an immunonutrient in aquaculture (Gupta et al., 2011). Levan when incorporated at 5 g/kg in the feeds of common carp C. carpio juveniles, provided protection from A. hydrophila (Rairakhwada et al., 2007). The same authors however noted that at 10 g/kg the prebiotic had an immunosuppressive effect on the fish. The juveniles of another carp variety, L. rohita were found to have high hemoglobin content, total leucocyte count, respiratory burst activity, serum lysozyme activity and highest relative survival percentage against A. hydrophila upon levan inclusion in the feed at 12.5 g/kg (Gupta et al., 2008). In a recent investigation, the same group demonstrated better temperature tolerance (increased levels of heat shock protein 70 in liver and muscle) in fish receiving similar amounts of dietary levan (Gupta et al., 2010). Though some of the prebiotic products may have the capacity to positively influence the health of the farmed fish, the information available at this point is few and far between to upscale its use in the feed industry. The two reviews mentioned earlier (Merrifield et al., 2010; Ringø et al., 2010) suggest that additional research efforts are needed to fill the knowledge gap. 5.2.3. Probiotics Probiotics are microorganisms that are believed to contribute to the well-being of the host organism. In human and veterinary sciences considerable knowledge exists on the salubrious benefits and applications of these microorganisms. Over the last five decades, several definitions were employed to describe them (Lee, 2009) and from a nutritional angle an allencompassing definition has been put forth by FAO: “Live microorganisms which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2002). For a bacterial strain to be accepted as a commercial probiotic by the food/feed industry, extensive in vitro and in vivo evidences on their innocuousness, adaptability and suitability to the target microbial environment, functionality including their physiological interactions with the host, and endurance to the processing technology are necessary. There has been a steady growth in the application of probiotic organisms in aquaculture and one should not forget the dual role probiotics have here – besides its use as feed additive, it is employed to support the microbial ecosystem in farms and hatcheries. Considering this additional use in aquaculture a new definition has been formulated: “A probiotic is defined as a live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment” (Verschuere et al., 2000). The immense interest on probiotics generated among aquaculture researchers has also been partly due to the emphasis given by FAO

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to promote better health among farmed fish through alternative strategies and to avoid the use of antibiotics. Though the nutritional modulation of health through probiotics was not directly suggested in the FAO report (Subasinghe, 1997), this aspect has become a focal area of research over the past several years. Through dietary application of probiotics, the host organism may derive benefits in multiple ways, which are categorized as: modification of gut microbiota, modulation of immune response and assistance to metabolic functions (Collado, 2009). The changes in the gut environment resulting from the presence of supplemented beneficial microorganisms (e.g. lactic acid bacteria) include the production of inhibitory compounds responsible for antimicrobial activity, competition with potential pathogens for binding sites or stimulation of epithelial barrier function, augmentation of immune responses such as that of the regulatory cytokines, inhibition of virulence gene or protein expression in gastrointestinal pathogens, and improvement of gut structure and digestion (Corr et al., 2009; Merrifield et al., 2010). Thus the delivery of appropriate probiotic microorganisms through feeds may provide better growth, elevated health status and greater disease resistance, thereby making it reasonable to think that the application of these microorganisms is one of the best approaches to preventive health care in aquatic animals. This is gaining ground in the aquaculture industry and over the last 10 years several studies have vouched the potential benefits of probiotic organisms. These studies have been summarized in Table 2 with emphasis on the immune responses in relation to the variety of organisms tested. It is clear from the table that much of the research on in-feed application of probiotics has focused on Bacillus spp. and Lactobacillus spp. In general, their levels of incorporation range between 107 and 108 CFU/g feed; the application period being 2–4 weeks for enhanced immune response and improved survival. Besides the aforementioned bacteria Vibrio sp., Carnobacterium sp., Pediococcus sp., Pseudomonas sp. and Psychrobacter sp. are reported to have beneficial effects in different fishes. The two latter species have been investigated in Atlantic cod both for their nutritional and immunological influence (Lazado et al., 2010, in press). Pediococcus acidilactici has been found to improve the defence mechanisms in shrimp (Castex et al., 2009, 2010) and prevent bone malformations in fish (Aubin et al., 2005). In Europe, this probiotic bacterium is now licensed as a commercial feed additive. Thus we find that there is great scope of developing an array of beneficial microorganisms, essentially those derived from the host. However, this demands stringent studies that would verify the claimed benefits. Different reviews (Balcázar et al., 2006; Kesarcodi-Watson et al., 2008; Wang et al., 2008; Qi et al., 2009; Nayak, 2010) that have appeared during the recent years provide us with a comprehensive understanding on the role of probiotics in fish health, particularly the one by Nayak, which has included aspects related to dosing and feeding.

6. Preventive nutrition Our knowledge on the immune system of fish is fragmentary, with the exception of a few species. This is a limiting factor for any attempt to link immune functions to nutrient/additive intake. Further, not all components of the immune system would respond equally to a particular substance. Marginal deficiencies and nutrient imbalances would impact an optimal immune response. Very few attempts have been directed to examine the underlying mechanisms as influenced by a nutrient or its interactions with other nutrients/additives. The interaction between nutrition and immune system of fish involves myriad physiological processes occurring in different organs under different levels of regulation. Since feeds are a complex mixture that delivers various nutrients and beneficial substances to support multiple physiological responses (Panserat and Kaushik, 2010), an integrative approach is necessary to analyse them. Recent approaches to understand biological processes through gene expression, molecular interactions and the cellular environment using high-throughput experimental techniques would contribute to systematic build-up of our understanding of the immune system of prominent farmed fishes. Therefore, prior to advocating the application of feeds for specific health benefits, we need to have a better understanding of the immune mechanisms, in order to clearly explain the attributes linked to a nutrient or additive. This knowledge on the immune mechanisms should include how individual variations in the immune responses determine the susceptibility to infections. In addition, we need to know how specific nutrients and their nutrient interactions are influenced by phenotypic and genotypic variations in fish populations. Further, the convincing end point will be to know the extent to which a recorded immunoenhancement translates to better disease resistance. In future, these facts would help us to evaluate feed components more effectively in order to clarify their roles in maintaining good health. The concept of preventive nutrition will gain wider acceptance in the industry since healthy fish would be capable of resisting pathogens.

7. Perspectives Aquatic feeds have evolved over the last half a century and the feed industry is aiming to optimize the quality of their products and to develop alternate ingredients that meet the physiological requirements of the animal, besides having minimal environmental impact. As functional ingredients are being increasingly used to add value to feed, vigilance should be exercised on those that are labelled immune-modulators. Extensive and rigorous research is still needed to validate the benefits of such ingredients to each target fish species. As more tools and markers become available for the major fish species, it would be possible to have a fair assessment on the benefits of the ingredients. Through this knowledge it should be possible to implement preventive health care strategies based on nutritional principles for aquaculture operations.

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Table 2 Parameter-wise summary of the immune responses, support to intestinal microbiota and protection from diseases in different fishes upon dietary administration of probiotics. Fish

Probiotic organism, dose (CFU/g feed) and feeding duration

Effects (with respect to control fish in most cases)a Enhancedb

Lysozyme activity Nile tilapia1

Rainbow trout2 Olive flounder3

Rainbow trout4

Rainbow trout5 Rainbow trout6 Complement activity Gilthead seabream7

Olive flounder3 Rainbow trout5 Plasma immunoglobulin Rohu8 Rainbow trout5 Phagocytic activity Rainbow trout2 Gilthead seabream7

Olive flounder3 4

Rainbow trout Rainbow trout5 Gilthead seabream9

Rainbow trout6 Cytotoxicity Gilthead seabream7 Gilthead seabream9

Respiratory burst Rainbow trout2 Gilthead seabream7 Olive flounder3 Rohu8 Rainbow trout5 Gilthead seabream9 Rainbow trout6

Bacillus subtilis – 1 × 107 ; 1 & 2 months Lactobacillus acidophilus – 1 × 107 ; 1 & 2 months B. subtilis + L. acidophilus – 0.5 × 107 ; 1 & 2 months Bacillus sp. – 2 × 108 ; 14 days Aeromonas sobria – 2 × 108 ; 14 days Lactobacil – 2.45 × 108 ; 30 days Sporolac – 2.45 × 108 ; 30 days Lactobacil + Sporolac – 2.45 × 108 ; 30 days Aeromonas hydrophila – 1 × 107 ; 7 & 14 days Vibrio fluvialis – 1 × 107 ; 7 & 14 days Carnobacterium sp. – 1 × 107 ; 7 & 14 days Lactobacillus rhamnosus – Live spray 1 × 1011 ; 30 days L. rhamnosus – Freeze dried 1 × 1011 ; 30 days Kocuria SM1 – 1 × 108 ; 1–4 weeks Pdp 11 or 51 M6 (Vibrionaceae family) – heat-inactivated 1 × 108 each; 4 weeks Pdp 11 + 51 M6 – 0.5 × 108 ; 4 weeks Lactobacil, Lactobacil + Sporolac – 2.45 × 108 ; 30 days Sporolac – 2.45 × 108 ; 30 days L. rhamnosus – Live spray 1 × 1011 ; 30 days L. rhamnosus – Freeze dried 1 × 1011 ; 30 days Bacillus subtilis – 1 × 108 ; 60 days L. rhamnosus – Live spray 1 × 1011 ; 30 days L. rhamnosus – Freeze dried 1 × 1011 ; 30 days Bacillus sp. – 2 × 108 ; 14 days Pdp 11 – 1 × 108 ; 4 weeks 51 M6 – 1 × 108 ; 4 weeks Pdp 11 + 51 M6 – 0.5 × 108 ; 4 weeks Lactobacil or Lactobacil + Sporolac – 2.45 × 108 ; 30 days Sporolac – 2.45 × 108 ; 30 days A. hydrophila or V. fluvialis or Carnobacterium sp. L. rhamnosus – Live spray 1 × 1011 ; 30 days L. rhamnosus – Freeze dried 1 × 1011 ; 30 days Bacillus subtilis – 1 × 107 ; 1 – 3 weeks Lactobacillus delbrueckii ssp. lactis – 1 × 107 ; 1–3 weeks B. subtilis + L. delbrueckii ssp. lactis – 0.5 × 107 each; 1–3 weeks Kocuria SM1 – 1 × 108 ; 1–4 weeks 51 M6 or Pdp 11 + 51 M6 – 1 × 108 or 0.5 × 108 ; 4 weeks B. subtilis or L. delbrueckii ssp. lactis – 1 × 107 ; 1–3 weeks B. subtilis + L. delbrueckii ssp. lactis – 0.5 × 107 each; 1–3 weeks Bacillus sp. – 2 × 108 ; 14 days Pdp 11 or 51 M6 or Pdp 11 + 51 M6 – 1 × 108 or 0.5 × 108 ; 4 weeks Lactobacil or Lactobacil + Sporolac – 2.45 × 108 ; 30 days Sporolac – 2.45 × 108 ; 30 days

1st, 2nd month 1st, 2nd month 1st, 2nd month

2nd, 4th week 4th week 2nd, 4th week

2nd week

2nd, 4th week 2nd, 4th week 20th, 30th day 20th day

20th day

2nd week 3rd week 2nd, 3rd week 2nd, 4th week 2nd, 4th week 20th day 20th, 30th day 2nd, 3rd week 2nd week 2nd, 3rd week 2nd, 3rd, 4th week 3rd week 3rd week

2nd, 4th week 2nd, 4th week

B. subtilis – 1 × 108 ; 60 days L. rhamnosus – Live spray 1 × 1011 ; 30 days L. rhamnosus – Freeze dried 1 × 1011 ; 30 days B. subtilis or L. delbrueckii – 1 × 107 ; 1 – 3 weeks or B. subtilis + L. delbrueckii –0.5 × 107 each; 1–3 weeks Kocuria SM1 – 1 × 108 ; 1–4 weeks

Serum bactericidal activity B. subtilis or L. acidophilus – 1 × 107 ; 1 & 2 months Nile tilapia1 B. subtilis + L. acidophilus – 0.5 × 107 ; 1 & 2 months

Unalteredb

1st and 2nd month against Aeromonas hydrophila, Pseudomonas fluorescens and Streptococcus iniae

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Table 2 (Continued) Fish

Probiotic organism, dose (CFU/g feed) and feeding duration

Effects (with respect to control fish in most cases)a Enhancedb

Unalteredb

Improved survival

Support Microbiota establishment after antibiotic treatment Same as above Same as above

Intestinal microbiota Rainbow trout10

Senegalese sole11

Bacillus subtilis + Bacillus licheniformis – 6.17 × 107 ;10 weeks

Enterococcus faecium – 2.29 × 108 ; 10 weeks B. subtilis + B. licheniformis – 1.12 × 108 + E. faecium – 1.70 × 108 ; 10 weeks Pdp13 or Pdp 11 (Shewanella sp.) – 1 × 109 ; 2 months

Pdp13 – Higher effect compared to Pdp11

Pathogen challenge Nile tilapia1

Rainbow trout2

B. subtilis/L. acidophilus – 1 × 107 ; 1 & 2 months/B. subtilis + L. acidophilus – 0.5 × 107 ; 1 & 2 months B. subtilis/B. subtilis + L. acidophilus (dose & duration as above) B. subtilis/B. subtilis + L. acidophilus (dose & duration as above) Bacillus sp. – 2 × 108 ; 14 days

Senegalese sole11

A. sobria GC2 – 2 × 108 ; 14 days Lactobacil – 2.45 × 108 ; 30 days Sporolac – 2.45 × 108 ; 30 days Lactobacil + Sporolac – 2.45 × 108 ; 30 days A. hydrophila or V. fluvialis or Carnobacterium sp. – 1 × 107 ; 7 & 14 days Pdp11 – 1 × 109 ; 2 months

Rohu8 Rainbow trout6

Pdp13 – 1 × 109 ; 2 months B. subtilis – 1 × 108 ; 60 days Kocuria SM1 – 1 × 108 ; 1–4 weeks

Olive flounder3 Olive flounder3 Rainbow trout4

2nd month – A. hydrophila (43–52%) 2nd month – P. fluorescens (33–51%) 2nd month – S. iniae (27–47%) A. salmonicida, Lactococcus garvieae, S. iniae, Vibrio anguillarum, V. ordalii, Yersinia ruckeri (87–100%) Same as above; (94–100%) Lymphocystic disease virus (70%) Same as above (55%) Same as above (75%) A. salmonicida (improved survival) Photobacterium damselae ssp. piscicida (25–30%) Same as above (30–35%) Edwardsiella tarda (75%) V. anguillarum – (2 week feeding 84%; 4 week 78%)

1 Oreochromis niloticus (Aly et al., 2008); 2 Oncorhynchus mykiss (Brunt et al., 2007); 3 Paralichthys olivaceus (Harikrishnan et al., 2010); 4 O. mykiss (Irianto and Austin, 2002); 5 O. mykiss (Panigrahi et al., 2005); 6 O. mykiss (Sharifuzzaman and Austin, 2009); 7 Sparus aurata (Díaz-Rosales et al., 2006); 8 Labeo rohita (Nayak et al., 2007); 9 S. aurata (Salinas et al., 2005); 10 O. mykiss (Merrifield et al., 2009); 11 Solea senegalensis (García de La Banda et al., 2010). a Grey shading suggests a response. b Time points mentioned under the columns ‘enhanced’ or ‘unaltered’ for the innate immune factors are observation points after offering the probiotic.

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