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Keywords:

  • innate immunity;
  • gut microbiota;
  • probiotics;
  • fish

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Innate immune system
  5. Integration of the immune response – cytokines
  6. Influence of gut microbiota on the health of fish
  7. Selecting probiotic strains
  8. Concluding remarks
  9. Acknowledgements
  10. References

Although fish immunology has progressed in the last few years, the contribution of the normal endogenous microbiota to the overall health status has been so far underestimated. In this context, the establishment of a normal or protective microbiota constitutes a key component to maintain good health, through competitive exclusion mechanisms, and has implications for the development and maturation of the immune system. The normal microbiota influences the innate immune system, which is of vital importance for the disease resistance of fish and is divided into physical barriers, humoral and cellular components. Innate humoral parameters include antimicrobial peptides, lysozyme, complement components, transferrin, pentraxins, lectins, antiproteases and natural antibodies, whereas nonspecific cytotoxic cells and phagocytes (monocytes/macrophages and neutrophils) constitute innate cellular immune effectors. Cytokines are an integral component of the adaptive and innate immune response, particularly IL-1β, interferon, tumor necrosis factor-α, transforming growth factor-β and several chemokines regulate innate immunity. This review covers the innate immune mechanisms of protection against pathogens, in relation with the installation and composition of the normal endogenous microbiota in fish and its role on health. Knowledge of such interaction may offer novel and useful means designing adequate therapeutic strategies for disease prevention and treatment.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Innate immune system
  5. Integration of the immune response – cytokines
  6. Influence of gut microbiota on the health of fish
  7. Selecting probiotic strains
  8. Concluding remarks
  9. Acknowledgements
  10. References

The health status of aquatic organisms is uniquely related to their immediate environments, which can contain very high concentrations of microorganisms. Many of these microorganisms are saprophytic, some are pathogenic and both types are capable of infecting fish when conditions become favorable for multiplication. However, under normal conditions fish maintain a healthy status by defending themselves against these potential invaders using a repertoire of innate and specific defense mechanisms (Ellis, 2001).

The immune systems of fish and higher vertebrates are similar and both have two integral components: (1) the innate, natural or nonspecific defense system formed by a series of cellular and humoral components, and (2) the adaptive, acquired or specific immune system characterized by the humoral immune response through the production of antibodies and by the cellular immune response, which is mediated by T-lymphocytes, capable of reacting specifically with antigens.

The innate immune system, unlike the specific immune system, lacks the ability to acquire memory and specific recognition after an encounter with foreign agents. However, this system is quite important in fish since the synthesis of antibodies is relatively slow in comparison with antibody production in the higher vertebrates. An adaptive immune response in ectothermic vertebrates takes considerable time (e.g., antibody production in salmonids takes at least 4–6 weeks) to respond and is very temperature-dependent (Ellis, 2001).

The main function of the innate immune system, i.e., the innate immune reactions mediated by monocytes/macrophages, comprises antigen presentation and regulation of the functional balance of immune response related to cytokine and chemokine receptor profiles. Although the host has evolved various tolerogenic mechanisms allowing a peaceful and productive coexistence with its normal endogenous microbiota, it remains highly responsive to enteropathogenic bacteria. This discriminatory ability represents a pivotal feature of efficient tolerance and homeostatic mechanisms.

Recently, the use of gnotobiotic animals has shown that bacteria have a profound impact on the anatomical, physiological and immunological development of the host (Rawls et al., 2004). Thus, establishing a healthy microbiota plays an important role in the generation of immunophysiologic regulation in the host by providing crucial signals for the development and maintenance of the immune system (Salminen et al., 2005).

Therefore, the focus of this review will be primarily on innate immune mechanisms of protection against pathogens as well as on the composition of the gut microbiota in fish, and particularly its role in maintaining health of the host.

Innate immune system

  1. Top of page
  2. Abstract
  3. Introduction
  4. Innate immune system
  5. Integration of the immune response – cytokines
  6. Influence of gut microbiota on the health of fish
  7. Selecting probiotic strains
  8. Concluding remarks
  9. Acknowledgements
  10. References

The primary line of defense in fish is the skin and mucus membranes. However, when pathogenic microorganisms enter the host, cellular and humoral innate defense mechanisms are activated (Magnadóttir, 2006). The most important mechanism involved in this defense is phagocytic activity, which will be described in detail later.

Epithelial barriers

Physical and chemical barriers, such as the dermis, epidermis, scales and mucus, constitute the first line of defense against disease-causing microorganisms in fish. The epidermal cells are capable of reacting against different aggressors and the integrity of these cells is fundamental to maintaining osmotic equilibrium, as well as impeding the entrance of foreign agents (Shephard, 1994).

Mucus, composed mainly of glycoprotein, prevents the colonization of foreign agents. The continuously maintained mucus layer provides a substrate in which the antibacterial mechanisms can occur by virtue of biologically active components including antibodies, antibacterial peptides, lysozymes, complement proteins, lectins and pentraxins (Nowak, 1999; Nagashima et al., 2001; Hellio et al., 2002).

Innate humoral immunity

The body fluids of the fish contain proteins and peptides that react against a great variety of microorganisms and microbial products. These nitrogenous compounds form part of the defense of the innate humoral immunity, and consist of antimicrobial peptides, lysozyme, complement, transferrin, pentraxins, lectins and antiproteases (Ellis, 1989).

Antimicrobial peptides (AMPs)

AMPs are present in tissues exposed to microorganisms such as mucosal surfaces and skin (Cole et al., 1997) and immune cells such as mast cells (Silphaduang & Noga, 2001; Murray et al., 2003). One type of AMPs expressed by fish mast cells (also known as eosinophilic granule cells) is piscidin, which has potent, broad-spectrum antibacterial activity against fish pathogens (Silphaduang & Noga, 2001).

Recently, other AMPs present in gill mast cells have been identified such as chrysophsin and pleurocidin, which have been isolated from red sea bream (Chrysophrys major) and winter flounder (Pleuronectes americanus), respectively (Iijima et al., 2003; Murray et al., 2003).

Lysozyme

Lysozyme is a cationic enzyme widely distributed in the serum, mucus, kidney, spleen and intestine of the fish (Lie et al., 1989). This enzyme is primarily associated with and synthesized by monocytes–macrophages and neutrophils (Murray & Fletcher, 1976; Nathan, 1987).

Lysozyme has the capacity to hydrolyze the chemical bond between the N-acetylmuramic acid and N-acetylglucosamine present in the peptidoglycan of bacterial cell walls. Lysozyme is able to lyse certain Gram-positive bacteria and, in conjunction with complement, even some Gram-negative bacteria (Paulsen et al., 2001).

Complement

The complement system comprises more than 35 soluble plasma proteins that are key to innate and adaptive immunity. Activation of the complement system initiates a cascade of biochemical reactions accompanied by the generation of biologically active mediators that result in antigen elimination via cell membrane lysis and activation of nonspecific mediators of inflammation (Holland & Lambris, 2002). There are three pathways that can activate the complement system: the classical pathway, which requires the presence of the antigen–antibody complex; the lectin pathway, which depends on the interaction of lectins such as mannose-binding lectin and ficolins with sugar moieties found on the surface of microorganisms, and finally the alternative pathway, which is activated directly by viruses, bacteria, fungi or even tumor cells and is independent of antibody (Boshra et al., 2006).

Transferrin

Transferrin, a bi-lobed monomeric glycoprotein, is responsible for the transport and delivery of iron to cells. Binding of iron to transferrin creates a bacteriostatic environment by limiting the availability of iron to replicating pathogens. Transferrin is also an acute phase protein invoked during an inflammatory response to remove iron from damaged tissue (Bayne & Gerwick, 2001) and also functions as an activator of fish macrophages (Stafford & Belosevic, 2003).

Interferon

Interferons (IFNs) are secreted by host cells, including macrophages, lymphocytes, natural killer cells and fibroblasts, in response to recognition of viral double-stranded RNA intermediates (Haller et al., 2006).

Two families of interferons can be distinguished on the basis of gene sequences, protein structure and functional properties. Type I IFNs, represented by the IFN-α and the IFN-β, which have a very similar biological activity. The IFN-α is synthesized mainly by the leukocytes and IFN-β by fibroblasts. Both types of interferons are produced in response to viral infections.

Type II IFN, as represented by IFN-γ, is produced by natural killer cells and T-lymphocytes in response to IL-12, IL-18, mitogens or antigens (Robertsen, 2006). In contrast to type I IFNs, IFN-γ is a key activator of macrophages for increased killing of bacterial, protozoal and viral pathogens.

Pentraxins: C-reactive protein (CRP) and serum amyloid protein (SAP)

Both CRP and SAP belong to a family of pentameric proteins called the pentraxins that bind their ligands in a calcium-dependent manner. They are commonly associated with the acute phase response.

CRP was discovered and named because of its reactivity with the phosphorylcholine residues of C-polysaccharide, the teichoic acid of Streptococcus pneumoniae (Tillett & Francis, 1930). The main biologic function of CRP is the ability to recognize pathogens and damaged cells of the host and to mediate their elimination by recruiting the complement system and phagocytic cells (Volanakis, 2001). In rainbow trout, CRP has showed opsonic activity for head kidney cells, resulting in enhanced phagocytic and chemokinetic activities (Kodama et al., 1999).

CRP is distinguished from SAP by its binding affinity for phosphorylcholine and phosphorylethanolamine. SAP only binds to phosphory-ethanolamine and can be purified as a result of its affinity for agarose.

Lectins

Lectins are usually constitutive proteins or glycoproteins, which possess binding activity towards carbohydrate residues. They have been grouped into classes based on the nature of their carbohydrate ligands, the biological processes in which they participate, their subcellular localization and their dependence on divalent cations (Drickamer & Taylor, 1993). A mannose-binding lectin, isolated from the serum of Atlantic salmon, has been shown to have opsonizing activity for a virulent strain of Aeromonas salmonicida (Ottinger et al., 1999).

Antiproteases

These antienzymes are characterized by their capacity to inhibit the action of proteases that some microorganisms utilize to penetrate the host. In teleost fish, an analogous protein to α1-antitrypsin was demonstrated (Hjelmeland, 1983). Another protein, which was demonstrated as homologous to α2-macroglobulin (Starkey et al., 1982), was reportedly capable of inhibiting several types of proteinases, including serine-, cysteine-, aspartic- and metallo-proteinases (Alexander & Ingram, 1992).

In addition, it has been observed that α2-macroglobulin present in the serum of rainbow trout is capable of inhibiting A. salmonicida protease (Ellis, 1987). The combined action of antithrombin and α2-macroglobulin in the plasma of Atlantic salmon was reported to inhibit the action of a serine protease of A. salmonicida (Salte et al., 1992). The differences in the α2-macroglobulin activity between the species of rainbow trout and brook trout have been directly correlated with their differing resistance to the infection caused by A. salmonicida (Freedman, 1991).

Natural antibodies (NA)

NA are secreted by B-cells without prior antigen-specific activation or antigen-driven selection. A large proportion of NA is polyreactive to phylogenetically conserved structures, such as nucleic acids, heat shock proteins, carbohydrates and phospholipids (Boes, 2000). The importance of NA functions in fish may be even greater than for higher vertebrates given that fish have neither appreciable affinity maturation responses nor class switch capabilities (Magor & Magor, 2001).

Recently, Sinyakov et al. (2002) observed that NA in the serum of goldfish (Carassius auratus) can be directly involved in the first line of resistance against A. salmonicida infection. In addition, these authors indicated that NA also may influence the level of antibody response since only the low NA carriers were capable of developing effective antibody response, and vice versa, the high NA carriers did not possess potential for active immunization.

Innate cellular immunity

The adaptive immunity effector function is mediated by T-lymphocytes, whereas nonspecific cytotoxic cells and phagocytes (monocytes/macrophages and neutrophils) constitute innate cellular immune effectors.

Nonspecific cytotoxic cells (NCC)

The NCC perform functions very similar to those of the higher vertebrates, acting on a wide variety of target cells, including allogeneic and xenogeneic tumor cells, virus-infected cells and protozoan parasites. NCC may also participate in antibacterial immunity by eliciting cytokine production and secretion (Jaso-Friedmann et al., 2001).

Phagocytosis

The innate cellular immune system is formed by a series of cells with essential functions to the host survival. Among these cells are the phagocytic cells, monocytes/macrophages and neutrophils, which play a fundamental role in protection and survival during adverse conditions. For example, antibody production is slow when there is a drop in temperature, therefore the host defense will depend almost exclusively on the phagocytic capacity.

Phagocytosis occurs when foreign objects such as bacteria adhere to the surface of the phagocyte, mediated by hydrophobic interactions or sugar/lectin interactions (Secombes, 1996). However, the most active promoter of phagocytosis is the C3 component of complement, which is bound to the bacterial surface lipopolysaccharide directly via the alternative pathway or indirectly via lectin or CRP (Ellis, 2001).

Antimicrobial response of fish phagocytes

Fish macrophages and neutrophils produce bactericidal reactive oxygen species (ROS) during the respiratory burst on contact with the particles or during phagocytosis or upon stimulation with a variety of agents. This process involves reduction of oxygen (O2) to the anionic radical superoxide (O2), which is catalyzed by an NADPH oxidase localized in the plasma and phagosomal membranes. Production of superoxide anion (O2) results in the spontaneous or enzyme-catalyzed production of an array of reactive oxygen products including hydrogen peroxide (H2O2), hydroxyl radical (OH·), hypochlorous acid (OCl) and peroxynitrite (ONOO), which have potent antimicrobial effects.

Production of nitric oxide (NO) constitutes another bactericidal mechanism, which is catalyzed by a NO synthase. Schoor & Plumb (1994) demonstrated inducible NO production, using enzyme histochemical techniques, from the anterior kidney of channel catfish (Ictalurus punctatus) infected with Edwardsiella ictaluri. Recently, Stafford et al. (2001) have characterized the molecules present in crude leukocyte supernatants that induce NO production in goldfish macrophages, suggesting that transferrin appears to be an important mediator for the activation of both fish macrophages and granulocytes.

Integration of the immune response – cytokines

  1. Top of page
  2. Abstract
  3. Introduction
  4. Innate immune system
  5. Integration of the immune response – cytokines
  6. Influence of gut microbiota on the health of fish
  7. Selecting probiotic strains
  8. Concluding remarks
  9. Acknowledgements
  10. References

Communication within the acquired immune system and between the innate and acquired systems is brought about by direct cell-to-cell contact involving adhesion molecules and by the production of chemical messengers. Chief among these chemical messengers are proteins called cytokines, which can induce a broad range of activities via multiple target cell types and their redundancy, indicated by the overlap in activities among different cytokines (Engelsma et al., 2002).

There are three functional categories of cytokines: (1) cytokines that regulate innate immune response; (2) cytokines that regulate adaptive immune response; and (3) cytokines that stimulate hematopoiesis.

Cytokines that regulate innate immunity are produced primarily by macrophages although they can also be produced by lymphocytes, NCC and other cells. They are produced in response to microbial antigens or compounds released from damaged cells. Among the mediators of inflammation released by activated phagocytes are the cytokines, particularly IL-1β, an important pro-inflammatory cytokine, interferon, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β) and several chemokines.

TNF-α is one of the principal mediators of the inflammatory response in mammals, transducing differential signals that regulate cellular activation and proliferation, cytotoxicity and apoptosis. When an inflammatory response is induced, the cascade of cytokine secretion begins with the release of TNF-α. This stimulates the release of IL-1β, which is then followed by the release of IL-6. The initiation of inflammation leads to the release of a myriad of other cytokines, which include chemoattractants that signal neutrophils and macrophages to migrate to the site of infection (e.g. chemokines).

Influence of gut microbiota on the health of fish

  1. Top of page
  2. Abstract
  3. Introduction
  4. Innate immune system
  5. Integration of the immune response – cytokines
  6. Influence of gut microbiota on the health of fish
  7. Selecting probiotic strains
  8. Concluding remarks
  9. Acknowledgements
  10. References

As has been indicated previously, fish health status is dependent on or conditioned to the immediate environment, since they are intimately in contact with a wide variety of microorganisms, including pathogenic and opportunistic bacteria that may colonize the external and internal body surfaces (Ellis, 2001). Thus, the establishment of a normal or protective microbiota is a key component in excluding potential invaders and maintaining health (Balcázar et al., 2006a). This is accomplished through competitive exclusion mechanisms and facilitates immune system development and maturation.

Colonization of the gastrointestinal tract of fish larvae starts immediately after hatching and is completed within a few hours. Colonizing bacteria can modulate expression of genes in the digestive tract, thus creating a favorable habitat for themselves and preventing invasion by other bacteria introduced later into the ecosystem (Balcázar et al., 2006b).

Traditionally, the influences of microbiota on the fish host have been obtained from comparisons of the physiological characteristics of germfree and conventional fish, but comparative research of this type can now be performed at the genomic level. The potential for obtaining exciting knowledge of mechanistic influences of the microbiota on the host by this approach has been demonstrated by the pioneering work of Rawls & colleagues (2004), who studied the effect of colonization by components of the microbiota in zebrafish (Danio rerio). Some genes were always expressed, independent of the type of bacteria used, while the expression of other genes was bacteria-specific, suggesting that at least a subset of zebrafish genes is sensitive to unknown factors induced by specific bacteria present in the gut microbiota.

Composition of gut microbiota

The relatively recent introduction of molecular techniques for the detection and quantification of microorganisms has led to a greater understanding of microbial diversity and its role in nature. Several studies involving molecular techniques have demonstrated that bacteria are the main constituent of the gut microbiota in fish (Spanggaard et al., 2000; Pond et al., 2006). However, some authors have also reported the presence of yeast (Andlid et al., 1998; Gatesoupe, 2007).

Although the composition of endogenous microbiota depends on genetic, nutritional and environmental factors, it is generally accepted that Gram-negative facultative anaerobic bacteria such as Acinetobacter, Alteromonas, Aeromonas, Flavobacterium/Cytophaga, Micrococcus, Moraxella, Pseudomonas and Vibrio constitute the predominant endogenous microbiota of a variety of species of marine fish (Cahill, 1990; Onarheim et al., 1994; Blanch et al., 1997). In contrast to saltwater fish, the endogenous microbiota of freshwater fish species tends to be dominated by members of the genera Aeromonas, Acinetobacter, Pseudomonas, Flavobacterium, representatives of the family Enterobacteriaceae, and obligate anaerobic bacteria of the genera Bacteroides, Clostridium and Fusobacterium (Sakata, 1990; Huber et al., 2004; Kim et al., 2007). In addition, various species of lactic acid bacteria have also been demonstrated to comprise part of this microbiota (Ringø & Gatesoupe, 1998; Balcázar et al., 2007a).

Immunity to bacterial pathogens

The external surface of fish is covered by a mucus layer, which acts as a medium for biologically active molecules (e.g. antibacterial peptides, lysozyme, lectins and proteases), and functions as the primary barrier to the adhesion and penetration of bacterial pathogens. Moreover, the gastrointestinal tract contains a diverse and complex endogenous microbiota, acids, bile salts and enzymes that can create a hostile environment for many pathogens. In most cases these properties are sufficient to protect against bacterial pathogens, which often only produce disease when conditions become favorable for their multiplication. If bacterial pathogens can breach these early lines of defense, cellular and humoral mechanisms are activated for preventing further spread of the infection. The complement system plays an essential role in alerting the host of the presence of microbial pathogens, as well as in their clearing. Complement can be activated directly by ‘foreign’ surfaces and also indirectly by other factors, principally CRP and lectin. Plasma also contains a number of soluble factors like antibacterial peptides, proteases and acute-phase proteins (pentraxins, transferrin, α2-macroglobulin, complement component C3, lysozyme and lectins). At the same time the cellular component of innate immunity is activated upon the recognition of pathogen-derived pathogen-associated molecular patterns, including lipopolysaccharide and double-stranded RNA as well as by host-derived cytokines. The latter group includes typical proinflammatory cytokines such as IL-1β, TNF-α and chemokines, which are of pivotal importance in recruiting monocytes/macrophages and neutrophils to the site of inflammation (Huising et al., 2003).

Probiotics as a strategy for improving health

The demonstration that the gut microbiota is an important component of mucosal barrier has resulted in the promotion of the use of beneficial probiotics. Probiotics have been defined by the World Health Organization-Food and Agriculture Organization as ‘live microorganisms which when administered in adequate amounts, confer a health benefit on the host’ (FAO/WHO, 2001).

Probiotic microorganisms consist mostly of strains of Bacillus (Salinas et al., 2005; Panigrahi et al., 2007), Carnobacterium (Robertson et al., 2000; Irianto & Austin, 2002; Kim & Austin, 2006a) and Lactobacillus (Nikoskelainen et al., 2001b; Panigrahi et al., 2004; Vendrell et al., 2007; Balcázar et al., 2007c), although the use of other species such as Aeromonas and Vibrio has also been explored (Austin et al., 1995; Irianto & Austin, 2002; Brunt & Austin, 2005).

Intake of probiotics has been demonstrated to modify the composition of the microbiota, and therefore assist in returning a disturbed microbiota (by antibiotics or other risk factors) to its normal beneficial composition. Mechanisms that may be implicated include the production of antimicrobial substances such as organic acids or bacteriocins (Balcázar et al., 2006c, 2007b), competition for nutrients or adhesion receptors (Nikoskelainen et al., 2001a; Vine et al., 2004; Balcázar et al., 2007b), inhibition of virulence gene expression (Defoirdt, 2007), and enhancement of the immune response (Nikoskelainen et al., 2003; Kim & Austin, 2006a; Balcázar et al., 2007c, d).

There is increasing evidence that probiotics enhance innate host resistance to microbial pathogens (Table 1). The findings of Irianto & Austin (2002) demonstrated that after feeding rainbow trout with probiotics containing Aeromonas hydrophila, Vibrio fluvialis, Carnobacterium sp. and Micrococcus luteus for 2 weeks, stimulation of humoral and cellular immunity was detected as demonstrated by an increase in lysozyme activity and in the number of erythrocytes, macrophages and lymphocytes. This finding offers an important example of the ability of nonpathogenic, endogenous microbial species to enhance the immunological functions of the host.

Table 1.   Probiotics used in fish and the effect on their host
Probiotic strainHost speciesEffectReference
Vibrio fluviales A3-47S, Aeromonas hydrophila A3-51, Carnobacterium sp. BA211, Micrococcus luteus A1-6Oncorhynchus mykissImmune stimulation and improved survival after challenge with Aeromonas salmonicidaIrianto & Austin (2002)
Lactobacillus rhamnosus ATCC 53103Oncorhynchus mykissImmune stimulation and improved survival after challenge with Aeromonas salmonicidaNikoskelainen et al. (2001b, 2003)
Lactococcus lactis CECT 539Scophthalmus maximusImmune stimulationVillamil et al. (2002)
Lactobacillus rhamnosus JCM 1136Oncorhynchus mykissImmune stimulationPanigrahi et al. (2004)
Lactobacillus delbriieckii CECT 287, Bacillus subtilis CECT 35Sparus aurataImmune stimulationSalinas et al. (2005)
Aeromonas sobria GC2Oncorhynchus mykissImmune stimulation and improved survival after challenge with Lactococcus garvieae and Streptococcus iniaeBrunt & Austin (2005)
Bacillus subtilis, Lactobacillus acidophilus, Clostridium butyricum, Saccharomyces cerevisiaeParalichthys olivaceusImmune stimulation and improved survival after challenge with Vibrio anguillarumTaoka et al. (2006)
Carnobacterium maltaromaticum B26 Carnobacterium divergens B33Oncorhynchus mykissImmune stimulation and improved survival after challenge with Aeromonas salmonicida and Yersinia ruckeri. Expression of cytokine genesKim & Austin (2006a, b)
Lactobacillus rhamnosus ATCC 53103Oreochromis niloticusImmune stimulation and improved survival after challenge with Edwardsiella tardaPirarat et al. (2006)
Lactobacillus rhamnosus ATCC 53103 Bacillus subtilis Enterococcus faeciumOncorhynchus mykissImmune stimulation and expression of cytokine genesPanigrahi et al. (2007)
Lactobacillus sakei CLFP 202, Lactococcus lactis CLFP 100 Leuconostoc mesenteroides CLFP 196Oncorhynchus mykiss, Salmo truttaImmune stimulation and improved survival after challenge with Aeromonas salmonicidaBalcázar et al. (2006d, 2007c, 2007d)
Lactobacillus plantarum CLFP 238 Leuconostoc mesenteroides CLFP 196Oncorhynchus mykissCompetitive exclusion and improved survival after challenge with Lactococcus garvieaeVendrell et al. (2007)

Probiotic strains have been shown to modulate the innate humoral responses and thereby facilitate the exclusion of potential pathogens. Panigrahi et al. (2004) fed rainbow trout a diet containing the probiotic Lactobacillus rhamnosus JCM1136. Evidence of an enhanced innate immune response was observed, including increased levels of serum lysozyme and complement activity. Similar observations have been described by Balcázar et al. (2007d), who demonstrated a positive effect on humoral immune response following probiotic administration (Lactococcus lactis ssp. lactis, Leuconostoc mesenteroides and Lactobacillus sakei) in brown trout (Salmo trutta).

An enhancement of phagocytic activity, which is responsible for early activation of the inflammatory response before antibody production, has also been reported in fish. Pirarat et al. (2006) demonstrated that after feeding tilapia (Oreochromis niloticus) with Lactobacillus rhamnosus ATCC 53103 for 2 weeks, stimulation of cellular immunity was detected as demonstrated by an increase in phagocytic activity. Similarly, Balcázar et al. (2006d) observed after feeding rainbow trout with probiotics containing Lactococcus lactis ssp. lactis, Leuconostoc mesenteroides and Lactobacillus sakei for 2 weeks, an enhanced phagocytosis of Aeromonas salmonicida by leukocytes isolated from mucosa-associated lymphoid tissues.

Probiotics can also modify the immune response of the host by interacting with epithelial cells and by modulating the secretion of anti-inflammatory cytokines, which could result in a reduction of inflammation. Recently, studies showed that IL-1β, IL-8, TNF-α, and TGF-β expression was not induced in rainbow trout gut cells following administration of the probiotic bacteria Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33. However, detection of significantly higher IL-1β and TNF-α expression in head kidney cells indicates induction of an anti-inflammatory effect (Kim & Austin, 2006b).

Selecting probiotic strains

  1. Top of page
  2. Abstract
  3. Introduction
  4. Innate immune system
  5. Integration of the immune response – cytokines
  6. Influence of gut microbiota on the health of fish
  7. Selecting probiotic strains
  8. Concluding remarks
  9. Acknowledgements
  10. References

To be a probiotic, a bacterial strain has to fulfil several criteria. Potential probiotics must be safe and free of plasmid-encoded antibiotic resistance genes, that could be passed to pathogenic organisms in the host. They must persist in the gastrointestinal tract long enough to elicit an effect. Ability to adhere and persist are also closely related to potential immune effects. They must have the ability to improve host health, they must be amenable to industrial processes necessary for commercial production and finally they must remain viable in the food product and during storage (Verschuere et al., 2000; Vine et al., 2006; Balcázar et al., 2006b).

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Innate immune system
  5. Integration of the immune response – cytokines
  6. Influence of gut microbiota on the health of fish
  7. Selecting probiotic strains
  8. Concluding remarks
  9. Acknowledgements
  10. References

The maintenance of a healthy status is complex and relies on a delicate balance between the immune system and the normal endogenous microbiota. The normal microbiota confers many benefits to the intestinal physiology of the host. Some of these benefits include the metabolism of nutrients and organic substrates, and the contribution of the phenomenon of colonization resistance. However, when this balance is upset, pathogens that arrive or that have already been present but in numbers too small to cause disease take the opportunity to multiply. The chemotherapeutic agents may also have a greater effect on the host normal microbiota than on the pathogens, thus upsetting the balance.

Therefore, probiotic supplementation can assist in returning a disturbed microbiota to its normal beneficial composition, and influence the fish immune response in different ways. They can induce the proportion of phagocytically active cells and the activation of complement receptor expression. They also can modulate the secretion of anti-inflammatory cytokines.

Understanding how the fish immune system generally responds to gut microbiota may be an important basis for targeting manipulation of the microbial composition. This might be of special interest to design adequate therapeutic strategies for disease prevention and treatment.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Innate immune system
  5. Integration of the immune response – cytokines
  6. Influence of gut microbiota on the health of fish
  7. Selecting probiotic strains
  8. Concluding remarks
  9. Acknowledgements
  10. References

J.L.B. was supported by a postdoctoral I3P contract from Consejo Superior de Investigaciones Científicas (CSIC). The authors thank J. Rhodes, C. Peter and L. Rivera for critical reading of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Innate immune system
  5. Integration of the immune response – cytokines
  6. Influence of gut microbiota on the health of fish
  7. Selecting probiotic strains
  8. Concluding remarks
  9. Acknowledgements
  10. References
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