MINIREVIEW

A review on the interactions between gut microbiota and innate immunity of sh

Geovanny D. Gomez1 & Jose Luis Balcazar2
1

Mariculture Research Laboratory, Ocean University of China, Qingdao, China; and 2Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Cientcas (CSIC), Eduardo Cabello, Vigo, Spain

Correspondence: Jose Luis Balcazar, Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Cientcas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain. Tel.: 134 986 214 457; fax: 134 986 292 762; e-mail: balcazar@iim.csic.es Received 20 April 2007; revised 12 September 2007; accepted 12 September 2007. First published online 17 December 2007. DOI:10.1111/j.1574-695X.2007.00343.x Editor: Willem van Leeuwen Keywords innate immunity; gut microbiota; probiotics; sh.

Abstract Although sh 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 inuences the innate immune system, which is of vital importance for the disease resistance of sh 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 nonspecic 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-1b, interferon, tumor necrosis factor-a, transforming growth factor-b 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 sh 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
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 sh when conditions become favorable for multiplication. However, under normal conditions sh maintain a healthy status by defending themselves against these potential invaders using a repertoire of innate and specic defense mechanisms (Ellis, 2001). The immune systems of sh and higher vertebrates are similar and both have two integral components: (1) the innate, natural or nonspecic defense system formed by a series of cellular and humoral components, and (2) the adaptive, acquired or specic immune system characterized by the humoral immune response through the production of antibodies and by the cellular immune response, which is
FEMS Immunol Med Microbiol 52 (2008) 145154

mediated by T-lymphocytes, capable of reacting specically with antigens. The innate immune system, unlike the specic immune system, lacks the ability to acquire memory and specic recognition after an encounter with foreign agents. However, this system is quite important in sh 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 46 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 proles. Although the host has evolved various tolerogenic mechanisms allowing
 c

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

146

G.D. Gomez & J.L. Balcazar

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 efcient 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 sh, and particularly its role in maintaining health of the host.

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 sh mast cells (also known as eosinophilic granule cells) is piscidin, which has potent, broad-spectrum antibacterial activity against sh pathogens (Silphaduang & Noga, 2001). Recently, other AMPs present in gill mast cells have been identied such as chrysophsin and pleurocidin, which have been isolated from red sea bream (Chrysophrys major) and winter ounder (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 sh (Lie et al., 1989). This enzyme is primarily associated with and synthesized by monocytesmacrophages 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).

Innate immune system

The primary line of defense in sh is the skin and mucus membranes. However, when pathogenic microorganisms enter the host, cellular and humoral innate defense mechan isms are activated (Magnadottir, 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 rst line of defense against disease-causing microorganisms in sh. 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). 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 nonspecic mediators of inammation (Holland & Lambris, 2002). There are three pathways that can activate the complement system: the classical pathway, which requires the presence of the antigenantibody complex; the lectin pathway, which depends on the interaction of lectins such as mannose-binding lectin and colins with sugar moieties found on the surface of microorganisms, and nally the alternative pathway, which is activated directly by viruses, bacteria, fungi or even tumor cells and is independent of antibody (Boshra et al., 2006).

Innate humoral immunity

The body uids of the sh 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).
 c

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
FEMS Immunol Med Microbiol 52 (2008) 145154

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

The role of gut microbiota in sh innate immunity

147

limiting the availability of iron to replicating pathogens. Transferrin is also an acute phase protein invoked during an inammatory response to remove iron from damaged tissue (Bayne & Gerwick, 2001) and also functions as an activator of sh macrophages (Stafford & Belosevic, 2003). Interferon Interferons (IFNs) are secreted by host cells, including macrophages, lymphocytes, natural killer cells and broblasts, 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-a and the IFN-b, which have a very similar biological activity. The IFN-a is synthesized mainly by the leukocytes and IFN-b by broblasts. Both types of interferons are produced in response to viral infections. Type II IFN, as represented by IFN-g, 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-g 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 afnity for phosphorylcholine and phosphorylethanolamine. SAP only binds to phosphory-ethanolamine and can be puried as a result of its afnity 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
FEMS Immunol Med Microbiol 52 (2008) 145154

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 sh, an analogous protein to a1-antitrypsin was demonstrated (Hjelmeland, 1983). Another protein, which was demonstrated as homologous to a2-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 a2-macroglobulin present in the serum of rainbow trout is capable of inhibiting A. salmonicida protease (Ellis, 1987). The combined action of antithrombin and a2-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 a2-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-specic 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 sh may be even greater than for higher vertebrates given that sh have neither appreciable afnity maturation responses nor class switch capabilities (Magor & Magor, 2001). Recently, Sinyakov et al. (2002) observed that NA in the serum of goldsh (Carassius auratus) can be directly involved in the rst line of resistance against A. salmonicida infection. In addition, these authors indicated that NA also may inuence 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 nonspecic cytotoxic cells and
 c

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

148

G.D. Gomez & J.L. Balcazar

phagocytes (monocytes/macrophages and neutrophils) constitute innate cellular immune effectors. Nonspecic 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, virusinfected 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 sh 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 (O), which is catalyzed by an NADPH oxidase localized in 2 the plasma and phagosomal membranes. Production of superoxide anion (O) results in the spontaneous or en2 zyme-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 catsh (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 goldsh macrophages, suggesting that trans c

ferrin appears to be an important mediator for the activation of both sh macrophages and granulocytes.

Integration of the immune response -- cytokines

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 inammation released by activated phagocytes are the cytokines, particularly IL-1b, an important pro-inammatory cytokine, interferon, tumor necrosis factor-a (TNF-a), transforming growth factor-b (TGF-b) and several chemokines. TNF-a is one of the principal mediators of the inammatory response in mammals, transducing differential signals that regulate cellular activation and proliferation, cytotoxicity and apoptosis. When an inammatory response is induced, the cascade of cytokine secretion begins with the release of TNF-a. This stimulates the release of IL-1b, which is then followed by the release of IL-6. The initiation of inammation 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

As has been indicated previously, sh 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 (Balcazar et al., 2006a). This is accomplished through competitive exclusion mechanisms and facilitates immune system development and maturation. Colonization of the gastrointestinal tract of sh larvae starts immediately after hatching and is completed within a few hours. Colonizing bacteria can modulate expression of
FEMS Immunol Med Microbiol 52 (2008) 145154

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

The role of gut microbiota in sh innate immunity

149

genes in the digestive tract, thus creating a favorable habitat for themselves and preventing invasion by other bacteria introduced later into the ecosystem (Balc zar et al., 2006b). a Traditionally, the inuences of microbiota on the sh host have been obtained from comparisons of the physiological characteristics of germfree and conventional sh, but comparative research of this type can now be performed at the genomic level. The potential for obtaining exciting knowledge of mechanistic inuences 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 zebrash (Danio rerio). Some genes were always expressed, independent of the type of bacteria used, while the expression of other genes was bacteria-specic, suggesting that at least a subset of zebrash genes is sensitive to unknown factors induced by specic bacteria present in the gut microbiota.

Composition of gut microbiota

The relatively recent introduction of molecular techniques for the detection and quantication 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 sh (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 sh (Cahill, 1990; Onarheim et al., 1994; Blanch et al., 1997). In contrast to saltwater sh, the endogenous microbiota of freshwater sh 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 (Ring & Gatesoupe, 1998; Balcazar et al., 2007a).

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 sufcient 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, a2-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 pathogenassociated molecular patterns, including lipopolysaccharide and double-stranded RNA as well as by host-derived cytokines. The latter group includes typical proinammatory cytokines such as IL-1b, TNF-a and chemokines, which are of pivotal importance in recruiting monocytes/macrophages and neutrophils to the site of inammation (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 benecial probiotics. Probiotics have been dened by the World Health Organization-Food and Agriculture Organization as live microorganisms which when administered in adequate amounts, confer a health benet 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; Balcazar 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 benecial composition. Mechanisms that may be implicated include the production of antimicrobial substances such as organic acids or bacteriocins (Balc zar et al., 2006c, 2007b), competition for a
 c

Immunity to bacterial pathogens

The external surface of sh is covered by a mucus layer, which acts as a medium for biologically active molecules (e.g. antibacterial peptides, lysozyme, lectins and proteases),
FEMS Immunol Med Microbiol 52 (2008) 145154

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

150

G.D. Gomez & J.L. Balcazar

nutrients or adhesion receptors (Nikoskelainen et al., 2001a; Vine et al., 2004; Balcazar et al., 2007b), inhibition of virulence gene expression (Defoirdt, 2007), and enhancement of the immune response (Nikoskelainen et al., 2003; Kim & Austin, 2006a; Balcazar et al., 2007c, d). There is increasing evidence that probiotics enhance innate host resistance to microbial pathogens (Table 1). The ndings of Irianto & Austin (2002) demonstrated that after feeding rainbow trout with probiotics containing Aeromonas hydrophila, Vibrio uvialis, 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 nding offers an important example of the ability of nonpathogenic, endogenous microbial species to enhance the immunological functions of the host. 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 Balc zar et al. (2007d), who demona strated 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 inammatory response before antibody production, has also been reported in sh. 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, Balcazar 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.

Table 1. Probiotics used in sh and the effect on their host Probiotic strain Vibrio uviales A3-47S, Aeromonas hydrophila A3-51, Carnobacterium sp. BA211, Micrococcus luteus A1-6 Lactobacillus rhamnosus ATCC 53103 Lactococcus lactis CECT 539 Lactobacillus rhamnosus JCM 1136 Lactobacillus delbriieckii CECT 287, Bacillus subtilis CECT 35 Aeromonas sobria GC2 Host species Oncorhynchus mykiss Effect Immune stimulation and improved survival after challenge with Aeromonas salmonicida Reference Irianto & Austin (2002)

Oncorhynchus mykiss Scophthalmus maximus Oncorhynchus mykiss Sparus aurata Oncorhynchus mykiss Paralichthys olivaceus Oncorhynchus mykiss Oreochromis niloticus Oncorhynchus mykiss Oncorhynchus mykiss, Salmo trutta Oncorhynchus mykiss

Immune stimulation and improved survival after challenge with Aeromonas salmonicida Immune stimulation Immune stimulation Immune stimulation Immune stimulation and improved survival after challenge with Lactococcus garvieae and Streptococcus iniae Immune stimulation and improved survival after challenge with Vibrio anguillarum Immune stimulation and improved survival after challenge with Aeromonas salmonicida and Yersinia ruckeri. Expression of cytokine genes Immune stimulation and improved survival after challenge with Edwardsiella tarda Immune stimulation and expression of cytokine genes Immune stimulation and improved survival after challenge with Aeromonas salmonicida Competitive exclusion and improved survival after challenge with Lactococcus garvieae

Nikoskelainen et al. (2001b, 2003) Villamil et al. (2002) Panigrahi et al. (2004) Salinas et al. (2005) Brunt & Austin (2005)

Bacillus subtilis, Lactobacillus acidophilus, Clostridium butyricum, Saccharomyces cerevisiae Carnobacterium maltaromaticum B26 Carnobacterium divergens B33 Lactobacillus rhamnosus ATCC 53103 Lactobacillus rhamnosus ATCC 53103 Bacillus subtilis Enterococcus faecium Lactobacillus sakei CLFP 202, Lactococcus lactis CLFP 100 Leuconostoc mesenteroides CLFP 196 Lactobacillus plantarum CLFP 238 Leuconostoc mesenteroides CLFP 196

Taoka et al. (2006)

Kim & Austin (2006a, b) Pirarat et al. (2006) Panigrahi et al. (2007)

Balcazar et al. (2006d, 2007c, 2007d) Vendrell et al. (2007)

 c

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

FEMS Immunol Med Microbiol 52 (2008) 145154

The role of gut microbiota in sh innate immunity

151

Probiotics can also modify the immune response of the host by interacting with epithelial cells and by modulating the secretion of anti-inammatory cytokines, which could result in a reduction of inammation. Recently, studies showed that IL-1b, IL-8, TNF-a, and TGF-b 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 signicantly higher IL-1b and TNF-a expression in head kidney cells indicates induction of an antiinammatory effect (Kim & Austin, 2006b).

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

References
Alexander JB & Ingram GA (1992) Noncellular nonspecic defence mechanisms of sh. Annu Rev Fish Dis 2: 249279. Andlid T, V zquez-Ju rez R & Gustafsson L (1998) Yeasts isolated a a from the intestine of rainbow trout adhere to and grow in intestinal mucus. Mol Mar Biol Biotechnol 7: 115126. Austin B, Stuckey LF, Robertson PAW, Effendi I & Grifth DRW (1995) A probiotic strain of Vibrio alginolyticus effective in reducing diseases caused by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalii. J Fish Dis 18: 9396. Balc zar JL, Decamp O, Vendrell D, de Blas I & Ruiz-Zarzuela I a (2006a) Health and nutritional properties of probiotics in sh and shellsh. Microb Ecol Health Dis 18: 6570. Balc zar JL, de Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell a D & Muzquiz JL (2006b) The role of probiotics in aquaculture. Vet Microbiol 114: 173186. Balc zar JL, de Blas I, Ruiz-Zarzuela I, Vendrell D, Evora MD & a Muzquiz JL (2006c) Growth inhibition of Aeromonas species by lactic acid bacteria isolated from salmonids. Microb Ecol Health Dis 18: 6163. Balc zar JL, Vendrell D, de Blas I, Ruiz-Zarzuela I, Giron s O & a e Muzquiz JL (2006d) Immune modulation by probiotic strains: quantication of phagocytosis of Aeromonas salmonicida by leukocytes isolated from gut of rainbow trout (Oncorhynchus mykiss) using a radiolabelling assay. Comp Immunol Microbiol Infect Dis 29: 335343. Balc zar JL, de Blas I, Ruiz-Zarzuela I, Vendrell D, Giron s O & a e Muzquiz JL (2007a) Sequencing of variable regions of the 16S rRNA gene for identication of lactic acid bacteria isolated from the intestinal microbiota of healthy salmonids. Comp Immunol Microbiol Infect Dis 30: 111118. Balc zar JL, Vendrell D, de Blas I, Ruiz-Zarzuela I, Giron s O & a e Muzquiz JL (2007b) In vitro competitive adhesion and production of antagonistic compounds by lactic acid bacteria against sh pathogens. Vet Microbiol 122: 373380. Balc zar JL, de Blas I, Ruiz-Zarzuela I, Vendrell D, Giron s O & a e Muzquiz JL (2007c) Enhancement of the immune response and protection induced by probiotic lactic acid bacteria against furunculosis in rainbow trout (Oncorhynchus mykiss). FEMS Immunol Med Microbiol 51: 185193. Balc zar JL, de Blas I, Ruiz-Zarzuela I, Vendrell D, Calvo AC, a M rquez I, Giron s O & Muzquiz JL (2007d) Changes in a e intestinal microbiota and humoral immune response following probiotic administration in brown trout (Salmo trutta). Br J Nutr 97: 522527. Bayne CJ & Gerwick L (2001) The acute phase response and innate immunity of sh. Dev Comp Immunol 25: 725743.

Selecting probiotic strains

To be a probiotic, a bacterial strain has to full 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 nally they must remain viable in the food product and during storage (Verschuere et al., 2000; Vine et al., 2006; Balc zar et al., 2006b). a

Concluding remarks
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 benets to the intestinal physiology of the host. Some of these benets 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 benecial composition, and inuence the sh 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 antiinammatory cytokines. Understanding how the sh 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.
FEMS Immunol Med Microbiol 52 (2008) 145154

 c

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

152

G.D. Gomez & J.L. Balcazar

Blanch AR, Alsina M, Simon M & Jofre J (1997) Determination of bacteria associated with reared turbot (Scophthalmus maximus) larvae. J Appl Microbiol 82: 729734. Boes M (2000) Role of natural and immune IgM antibodies in immune responses. Mol Immunol 37: 11411149. Boshra H, Li J & Sunyer JO (2006) Recent advances on the complement system of teleost sh. Fish Shellsh Immunol 20: 239262. Brunt J & Austin B (2005) Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 28: 693701. Cahill MM (1990) Bacterial ora of shes: a review. Microb Ecol 19: 2141. Cole AM, Weis P & Diamond G (1997) Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter ounder. J Biol Chem 272: 1200812013. Defoirdt T (2007) Quorum sensing disruption and the use of short-chain fatty acids and polyhydroxyalkanoates to control luminescent vibriosis. PhD Thesis, Ghent University, Belgium. Drickamer K & Taylor ME (1993) Biology of animal lectins. Annu Rev Cell Biol 9: 237264. Ellis AE (1987) Inhibition of the Aeromonas salmonicida extracellular protease by a2-macroglobulin in the serum of rainbow trout. Microb Pathog 3: 167177. Ellis AE (1989) The immunology of teleosts. Fish Pathology ` (Roberts RJ, ed), pp. 135152. Bailliere Tindall, London. Ellis AE (2001) Innate host defense mechanisms of sh against viruses and bacteria. Dev Comp Immunol 25: 827839. Engelsma MY, Huising MO, van Muiswinkel WB, Flik G, Kwang J, Savelkoul HFJ & Verburg-van Kemenade BML (2002) Neuroendocrine-immune interactions in sh: a role for interleukin-1. Vet Immunol Immunopathol 87: 467479. FAO/WHO (2001) Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria. Cordoba, Argentina. Freedman SJ (1991) The role of a2-macroglobulin in furunculosis: a comparison of rainbow trout and brook trout. Comp Biochem Physiol B 98: 549553. Gatesoupe FJ (2007) Live yeasts in the gut: natural occurrence, dietary introduction, and their effects on sh health and development. Aquaculture 267: 2030. Haller O, Kochs G & Weber F (2006) The interferon response circuit: induction and suppression by pathogenic viruses. Virology 344: 119130. Hellio C, Pons AM, Beaupoil C, Bourgougnon N & Le Gal Y (2002) Antibacterial, antifungal and cytotoxic activities of extracts from sh epidermis and epidermal mucus. Int J Antimicrob Agents 20: 214219. Hjelmeland K (1983) Proteinase inhibitors in the muscle and serum of cod (Gadus morhua). Isolation and characterization. Comp Biochem Physiol B 76: 365372. Holland MCH & Lambris JD (2002) The complement system in teleosts. Fish Shellsh Immunol 12: 399420.

Huber I, Spanggaard B, Appel KF, Rossen L, Nielsen T & Gram L (2004) Phylogenetic analysis and in situ identication of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum). J Appl Microbiol 96: 117132. Huising MO, Stolte E, Flik G, Savelkoul HFJ & Verburg-van Kemenada BML (2003) CXC chemokines and leukocyte chemotaxis in common carp (Cyprinus carpio L.). Dev Comp Immunol 27: 875888. Iijima N, Tanimoto N, Emoto Y, Morita Y, Uematsu K, Murakami T & Nakai T (2003) Purication and characterization of three isoforms of chrysophsin, a novel antimicrobial peptide in the gills of the red sea bream, Chrysophrys major. Eur J Biochem 270: 675686. Irianto A & Austin B (2002) Use of probiotics to control furunculosis in rainbow trout Oncorhynchus mykiss (Walbaum). J Fish Dis 25: 333342. Jaso-Friedmann L, Leary JH III & Evans DL (2001) The nonspecic cytotoxic cell receptor (NCCRP-1): molecular organization and signalling properties. Dev Comp Immunol 25: 701711. Kim DH & Austin B (2006a) Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics. Fish Shellsh Immunol 21: 513524. Kim DH & Austin B (2006b) Cytokine expression in leucocytes and gut cells of rainbow trout, Oncorhynchus mykiss Walbaum, induced by probiotics. Vet Immunol Immunopathol 114: 297304. Kim DH, Brunt J & Austin B (2007) Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss). J Appl Microbiol 102: 16541664. Kodama H, Arimitsu H, Mukamoto M & Sugimoto C (1999) Enhancement of phagocytic and chemokinetic activities of rainbow trout head kidney cells by C-reactive protein. Am J Vet Res 60: 240244. Lie , Evensen A, Srensen A & Frysadal E (1989) Study on lysozyme activity in some sh species. Dis Aquat Org 6: 15. Magnadottir B (2006) Innate immunity of sh (overview). Fish Shellsh Immunol 20: 137151. Magor BG & Magor KE (2001) Evolution of effectors and receptors of innate immunity. Dev Comp Immunol 25: 651682. Murray CK & Fletcher TC (1976) The immunohistochemical localization of lysozyme in plaice (Pleuronectes platessa L.) tissues. J Fish Biol 9: 329334. Murray HM, Gallant JW & Douglas SE (2003) Cellular localization of pleurocidin gene expression and synthesis in winter ounder gill using immunohistochemistry and in situ hybridization. Cell Tissue Res 312: 197202. Nagashima Y, Sendo A, Shimakura K, Shiomi K, Kobayashi T, Kimura B & Fujii T (2001) Antibacterial factors in skin mucus of rabbitshes. J Fish Biol 58: 17611765. Nathan CF (1987) Secretory product of macrophages. J Clin Invest 79: 319326.

 c

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

FEMS Immunol Med Microbiol 52 (2008) 145154

The role of gut microbiota in sh innate immunity

153

Nikoskelainen S, Salminen S, Bylund G & Ouwehand AC (2001a) Characterization of the properties of human and dairy-derived probiotics for prevention of infectious diseases in sh. Appl Environ Microbiol 67: 24302435. Nikoskelainen S, Ouwehand A, Salminen S & Bylund G (2001b) Protection of rainbow trout (Oncorhynchus mykiss) from furunculosis by Lactobacillus rhamnosus. Aquaculture 198: 229236. Nikoskelainen S, Ouwehand AC, Bylund G, Salminen S & Lilius EM (2003) Immune enhancement in rainbow trout (Oncorhynchus mykiss) by potential probiotic bacteria (Lactobacillus rhamnosus). Fish Shellsh Immunol 15: 443452. Nowak BF (1999) Signicance of environmental factors in aetiology of skin diseases of teleost sh. Bull Eur Assoc Fish Pathol 19: 290292. Onarheim AM, Wiik R, Burghardt J & Stackebrandt E (1994) Characterization and identication of two Vibrio species indigenous to the intestine of sh in cold sea water; description of Vibrio iliopiscarius sp. nov. Syst Appl Microbiol 17: 370379. Ottinger CA, Johnson SC, Ewart KV, Brown LL & Ross NW (1999) Enhancement of anti-Aeromonas salmonicida activity in Atlantic salmon (Salmo salar) macrophages by mannosebinding lectin. Comp Biochem Physiol C 123: 5359. Panigrahi A, Kiron V, Kobayashi T, Puangkaew J, Satoh S & Sugita H (2004) Immune responses in rainbow trout Oncorhynchus mykiss induced by a potential probiotic bacteria Lactobacillus rhamnosus JCM 1136. Vet Immunol Immunopathol 102: 379388. Panigrahi A, Kiron V, Satoh S, Hirono I, Kobayashi T, Sugita H, Puangkaew J & Aoki T (2007) Immune modulation and expression of cytokine genes in rainbow trout Oncorhynchus mykiss upon probiotic feeding. Dev Comp Immunol 31: 372382. Paulsen SM, Engstad RE & Robertsen B (2001) Enhanced lysozyme production in Atlantic salmon (Salmo salar L.) macrophages treated with yeast b-glucan and bacterial lipopolysaccharide. Fish Shellsh Immunol 11: 2337. Pirarat N, Kobayashi T, Katagiri T, Maita M & Endo M (2006) Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Vet Immunol Immunopathol 113: 339347. Pond MJ, Stone DM & Alderman DJ (2006) Comparison of conventional and molecular techniques to investigate the intestinal microora of rainbow trout (Oncorhynchus mykiss). Aquaculture 261: 194203. Rawls JF, Samuel BS & Gordon JI (2004) Gnotobiotic zebrash reveal evolutionarily conserved responses to the gut microbiota. Proc Natl Acad Sci USA 101: 45964601. Ring E & Gatesoupe FJ (1998) Lactic acid bacteria in sh: a review. Aquaculture 160: 177203. Robertsen B (2006) The interferon system of teleost sh. Fish Shellsh Immunol 20: 172191.

Robertson PAW, ODowd C, Burrells C, Williams P & Austin B (2000) Use of Carnobacterium sp. as a probiotic for Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss, Walbaum). Aquaculture 185: 235243. Sakata T (1990) Microora in the digestive tract of sh and shellsh. Microbiology in Poecilotherms (Lesel R, ed), pp. 171176. Elsevier, Amsterdam. Salinas I, Cuesta A, Esteban MA & Meseguer J (2005) Dietary administration of Lactobacillus delbriieckii and Bacillus subtilis, single or combined, on gilthead seabream cellular innate immune responses. Fish Shellsh Immunol 19: 6777. Salminen SJ, Gueimonde M & Isolauri E (2005) Probiotic that modify disease risk. J Nutr 135: 12941298. Salte R, Norberg K, Arnesen JA, degaard OR & Eggset G (1992) Serine protease and glycerophospholipid: cholesterol acyltransferase of Aeromonas salmonicida work in concert in thrombus formation; in vitro the process is counteracted by plasma antithrombin and a2-macroglobulin. J Fish Dis 15: 215227. Schoor WP & Plumb JA (1994) Induction of nitric oxide synthase in channel catsh (Ictalurus punctatus) by Edwardsiella ictaluri. Dis Aquat Org 19: 153155. Secombes CJ (1996) The non-specic immune system:cellular defenses. The sh immune system: organism, pathogen and environment (Iwama G & Nakanishi T, eds), pp. 63103. Academic Press, San Diego. Shephard KL (1994) Functions for sh mucus. Rev Fish Biol Fish 4: 401429. Silphaduang U & Noga EJ (2001) Peptide antibiotics in mast cells of sh. Nature 414: 268269. Sinyakov MS, Dror M, Zhevelev HM, Margel S & Avtalion RR (2002) Natural antibodies and their signicance in active immunization and protection against a dened pathogen in sh. Vaccine 20: 36683674. Spanggaard B, Huber I, Nielsen J, Nielsen T, Appel KF & Gram L (2000) The microora of rainbow trout intestine: a comparison of traditional and molecular identication. Aquaculture 182: 115. Stafford JL & Belosevic M (2003) Transferrin and the innate immune response of sh: identication of a novel mechanism of macrophage activation. Dev Comp Immunol 27: 539554. Stafford JL, Neumann NF & Belosevic M (2001) Products of proteolytic cleavage of transferring induce nitric oxide response of goldsh macrophages. Dev Comp Immunol 25: 101115. Starkey PM, Fletcher TC & Barrett AJ (1982) Evolution of a2-macroglobulin. The purication and characterization of a protein homologous with human a2 macroglobulin from plaice (Pleuronectes platessa L.). Biochem J 205: 97104. Taoka Y, Maeda H, Jo J-Y, Jeon M-J, Bai SC, Lee W-J, Yuge K & Koshio S (2006) Growth, stress tolerance and non-specic immune response of Japanese ounder Paralichthys olivaceus

FEMS Immunol Med Microbiol 52 (2008) 145154

 c

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

154

G.D. Gomez & J.L. Balcazar

to probiotics in a closed recirculating system. Fisheries Science 72: 310321. Tillett WS & Francis T (1930) Serological reactions in pneumonia with a non-protein somatic fraction of pneumococcus. J Exp Med 52: 561571. Vendrell D, Balc zar JL, de Blas I, Ruiz-Zarzuela I, Giron s O & a e Muzquiz JL (2007) Protection of rainbow trout (Oncorhynchus mykiss) from lactococcosis by probiotic bacteria. Comp Immunol Microbiol Infect Dis (in press). Verschuere L, Rombaut G, Sorgeloos P & Verstraete W (2000) Probiotic bacteria as biological control agents in aquaculture. Microbiol Mol Biol Rev 64: 655671.

Villamil L, Tafalla C, Figueras A & Novoa B (2002) Evaluation of immunomodulatory effects of lactic acid bacteria in turbot (Scophthalmus maximus). Clin Diagn Lab Immunol 9: 13181323. Vine NG, Leukes WD & Kaiser H (2004) In vitro growth characteristics of ve candidate aquaculture probiotics and two sh pathogens grown in sh intestinal mucus. FEMS Microbiol Lett 231: 145152. Vine NG, Leukes WD & Kaiser H (2006) Probiotics in marine larviculture. FEMS Microbiol Rev 30: 404427. Volanakis JE (2001) Human C-reactive protein: expresion, structure, and function. Mol Immunol 38: 189197.

 c

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

FEMS Immunol Med Microbiol 52 (2008) 145154