1. Introduction
The innate immune system is the only defence weapon of invertebrates and a fundamental defence mechanism of fish. The bony fishes are derived from one of the earliest divergent vertebrate lineages to have both innate and acquired immune systems. The innate system also plays an instructive role in the acquired immune response and homeostasis (Magnado´ttir, 2006).
The innate immune system of fish is divided into physical barriers, cellular and humoral components although there are some important differences when comparing with other vertebrates (Uribe et al., 2011). Fish are considered to be an ideal model to study the underpinnings of immune systems precisely because of their phylogenetic position and the fact that their adaptive immune systems have not been elaborated to the extent seen in mammals (Magor and Magor, 2001).
On the other hand, aquaculture industry demands better knowledge of immunity of farmed fish species. While most available studies focus on cellular and humoral components of fish immune system, less much attention has been focus on physical barriers. The vertebrates immune system includes primary (organs in which lymphopoiesis takes place) and secondary (places of interaction between the different immune cells that have as mission provide a favorable environment for developing the immune response) lymphoid organs.
One of the secondary organs is the MALT (mucous associated lymphoid tissue), which is the major lymphoid structure of the body. Since the majority of the infectious agents affects or initiates his process of infection in the mucous surfaces, the mucosal immune response plays a crucial role in the course of the infection (McNeilly et al., 2008). Skin, gill and gut are part of the fish mucosal immunity and constitute a large area (much greater than that of other vertebrates) for the possible invasion of pathogens. Among them particular interest (given its extension) has the GALT (Maaser and Kagnoff, 2002) while two very special fish organ, skin and gills, as organs implied in the first barrier to infection remains largely unknown (Ingram, 1980; Shephard, 1994; Ellis, 2001).
The layers of tegument of teleosts are the cuticle o mucus layer (with a very complex composition), which have bacteria forming the microbiota, the epidermis (scamous stratified epithelium with goblet cells) and dermis (with two layers, the hypodermis or stratum spongiosum, a frequent site of development of infectious processes and the innermost layer or stratum compactum) (Esteban, 2012). In fish the epidermal mucus is considered a key component of innate immunity it is produced primarily by epidermal goblet cells or mucus cells and is composed of water and gel-forming macromolecules including mucins and other glycoproteins (Ingram, 1980).
Research on fish mucus has been undertaken for many decades. The origins of fish mucus, as well as the composition of different types of fish mucus have been well identified by many investigations using histological and biochemical techniques (Kerry, 1994; Al-Arifa et al., 2011; Bragadeeswaran et al., 2011). Conclusions of these studies show that the mucus insures many important biological functions that allow fishes to survive and to adapt to their environment (Balebona et al., 1995; Al-Arifa et al., 2011). Recently, and using more precise and sophisticated techniques, many studies were reinterested on fish mucus for many objectives.
Some studies tend to better understand the composition of fish mucus, their functions, and the effect of environmental circumstances on the mucus secretion (Al-Arifa et al., 2011), while others focused on biotechnological purposes such as possible uses of mucus in many other domains, such as to ameliorate conditions of fish cultures in farms (Hellio et al., 2002; Blanco et al., 2007).
The fish skin mucus is considered as a natural, physical, biochemical, dynamic, and semipermeable barrier that enables the exchange of nutrients, water, gases, odorants, hormones, and gametes (Esteban, 2012). Many fish species locally secrete adhesive mucus which is important for successful substrate attachment (Thomas and Hermans, 1985), whereas others may secrete antiadhesive mucus, which prevents colonization by fouling organisms (McKenzie and Grigovala, 1996).
Mucus secretions may also contain toxins and lytic compounds to deter fouling organisms or predators (Bavington et al., 2004). Mucus is considered predominantly on the general skin surface and gills, but also from the gut lining; indeed on all epidermal surfaces that mark the interface between the fish and external environment. Skin mucus can trap and immobilize pathogens before they can contact epithelial surfaces, because it is impermeant to most bacteria and many pathogens (Mayer, 2003; Cone, 2009). Furthermore, mucus is continuously secreted and replaced, which prevents the stable colonization of potential infectious microorganisms as well as invasion of metazoan parasites (Ingram, 1980; Ellis, 2001; Nagashima et al., 2003).
Sometimes the mucus layer can be shed or digested, thus pathogens must move ‘upstream’ through the unstirred layers of mucus adhering to the cells on the epithelium surface or penetrate a mucus ‘blanket’ before it is shed (Cone, 2009). Mucus is a complex fluid and its composition varies throughout the epithelial surface and among fish species. The composition and characteristics of skin mucus is very important for the maintenance of its immune functions (Cone, 2009). Furthermore, mucous cells and the compositions of the mucus they produce are influenced by endogenous factors (e.g. sex, developmental stage) and exogenous factors (such as stress, acid and infections) (Blackstock and Pickering, 1982; Zaccone et al., 1985).
The skin, gills, and intestine of most fishes are covered by a gel-forming molecule called mucus; the mucus of fishes is the site of contact direct of fish with their environment. Many functions are attributed to fish mucus, being noticeable among them its important role in the ionic and osmotic regulation, feeding, attachment, defence, cleaning, respiration, communication and reproduction (Kerry, 1994; Bavington et al., 2004).
Furthermore, the fish mucus acts as the first barrier against an infection and it is the first site of interaction between fish cells and pathogens, being the first route for both aerobic and anaerobic bacteria to reach the fish cells. Thus, the mucus seems to have an important selective role to discriminate between pathogenic and commensal bacterial strains. Concomitantly, mucus plays a critical role in the defense mechanism of the fishes by also acting as a biological barrier (Raj et al., 2011; Subramanian et al., 2007, 2008; Esteban, 2012). The fish mucus composition as well as the immunological defenses that occurred in fish skin and mucus was detailed reviewed by Esteban (2012). In the present review, we will focus essentially in the interaction that occurred between fish skin mucus and pathogen bacteria.
2. Bacteria population of fish skin
Before 1980, the identification of bacteria related to fish refers only to the genera of bacteria isolated (Marian, 1990). Fortunately, using molecular techniques, numerical taxonomy and essentially the determination of Guaninecytosine percentage in DNA (%G + C), it became possible to attain an accurate identification of these bacterial strains. In fact, these molecular and numerical techniques are culture-independent and make possible scientists to even identify uncultured bacteria. Depending on the purpose of study, many researches investigated a comparison between phenotypic and molecular analyses (Spanggaard et al., 2000).
Nevertheless, numerical, molecular, and conventional approaches are still complementary to reach a precise identification of bacteria. Even if some scientists thought that narrow-based studies focusing on a limited number of bacterial groups are often more successful than those that attempt to be broad-based (Austin, 2006), it appears that using combination of numerical and conventional techniques could allow to identify the majority of the bacterial population related to fish. This identification would be useful, not only to identify the possible pathogen bacteria and help to choose the suitable treatment, but also to identify the beneficial bacteria that can improve fish status such as probiotics. Knowing that fish are in a very close contact with their environment, the bacterial genera present in the skin mucus seems to be those from the environment or diet and it is apparent that there are many similarities between the bacterial populations in fish and water (Apun et al., 1999; Diler et al., 2000; Austin, 2006).
Nevertheless, the bacterial population present in farmed fish changes depending on the storage conditions (temperature, pH, etc.), the diet, and the concentration of pollutants in water, and even on the season (Bernadsky and Rosenberg, 1992). These conditions will absolutely influence the microflora on external and internal surfaces (including the mucus, the skin, the gills and the digestive tract of fishes). Certainly, colonization may well start at the egg and/or larval stage, and continue with the development of the fish (Olafsen, 2001). Thus, the numbers and range of microorganisms present in the eggs, in food and in the water, will influence the microflora ofthe developing fish (Austin, 2006). The bacterial population colonizing internal and external surface also depend strongly on the health status of fishes, thus fish mucus can encompass benefit, opportunist and pathogen bacteria.
The bacterial population, including aerobic and anaerobic strains, in the skin of fish is estimated in gram-¹ of skin or in cm-2 of skin surface. However, this bacterial population depends on the fish environment, fish species and its health status. In fact, a population of 10² –10³ culturable bacteria cm of skin was enumerated in the Atlantic salmon (Salmo salar) in U.K., whereas rainbow trout (Oncorhynchus mykiss) from Turkey contained bacterial populations of 10¹ –10^7 g- ¹ (Diler et al., 2000) and freshly caught mullet (Mugil cephalus), whiting (Sillago ciliato), and flathead (Platycephalus fiscus) from Australia were reported to have seemingly higher populations of 4 * 10 ³ to 8 * 10⁴ bacteria cm- ² (Horsley, 1973; Gillespie and Macrae, 1975; Austin, 2006). Many pathogenic bacterial species were isolated from fish.
This population, made of different Gram shape, includes: (a) strict aerobic strains such as Acinetobacter johnsonii, Enterobacter aerogenes, Flavobacterium psychrophilum, Flexibacter spp., Micrococcus luteus, Moraxella spp., Pseudomonas fluorescens, Psychrobacters, Acinetobacter calcoaceticus, Alcaligenes faecalis; (b) aerobic–anaerobic facultative bacteria have also been isolated, this population belongs essentially to the genus Aeromonas (Aeromonas hydrophila, A. bestiarum, A. caviae, A. jandaei, A. schubertii, A. veronii biovar sobria), the genus Vibrio (Vibrio fluvialis, V. harveyi, V. albensis, V. anguillarum, V. splendidus biotype I, V. fischeri, V. ordalii, V. scophthalmi, V. alginolyticus) and the genus Photobacterium (P. damselae, P. damselae subsp. Piscicida, P. angustum, P. logei); (c) many other bacteria belonging to other genus have been isolated, such as Alcaligenes piechaudii, Escherichia coli, Bacillus cereus, B. firmus, Caulobacter, coryneforms, Cytophaga/ Flexibacter, Hyphomicrobium vulgare, Prosthecomicrobium, Pseudomonas marina, Alteromonas, Plesiomonas shigelloides, Moraxella, Neisseria, Yersinia ruckeri (Christensen, 1977; Allen et al., 1983; Montes et al., 1999; Diler et al., 2000; Gonzalez et al., 2000, 2001; Zmyslowska et al., 2001; Austin, 2006; Anja et al., 2000). Although the major bacterial species known to be pathogenic for fish are aerobic or aerobic–anaerobic facultative strains, some strict anaerobic bacteria were essentially isolated from fish intestine. In fact, the genus Clostridium was recovered from southern flounder (Paralichthys lethostigma), while both Clostridium and Gram negative genera including Fusobacterium, Bacteroides and Porphorymonas were recovered from Oscars (Astronotus ocellatus) and angelfish (Pterophyllum scalare) (Rachel and Beverly, 2003). Nevertheless, more detailed studies using sophisticated technics should be undertaken to know the eventual pathogenicity of other uncultivable bacterial strains. Moreover, it is difficult to define an exhaustive list of pathogenic bacteria due to the high possibility of transformation of bacteria to pathogenic strain. In fact, the gene transfer between bacteria is quite known and it can convert a previously avirulent strain into a virulent pathogen (Rachel and Beverly, 2003).
3. Pathogens adhesion mechanisms: implicated components and factors influencing the adhesion
To study the adhesion of beneficial or pathogen bacteria to fish, it seems important to understand the mechanisms of attachment of bacteria to mucus and skin, as well as the majors factors controlling this attachment. The initial attachment of bacterial cells to a surface (e.g. mucus) involve many factors including hydrophobicity, surface charge, surface roughness, surface micro-topography and water flow, components of the surface, pH of the milieu, viscosity, etc. (Corte´ s et al., 2011). The mechanism of bacterial attaching to surfaces follows an organized sequence starting with the deposition of specific adhesive protein which binds to the surface reversibly. A successive deposition of cells creates a strong binding by cell to cell cohesion and cell-binding-proteins. Cell adhesion molecules involved in the process are first hydrolyzed by extracellular enzymes (Pavithra and Doble, 2008; Corte´ s et al., 2011). Bacterial adhesion is directly related to protein adsorption. Some studies demonstrated that the bacterial attachment to surfaces occurs essentially in two-step (Kumar and Anand, 1998; Marshall et al., 1971). In the first step, the bacteria must be transported close enough to allow initial attachment to take place. Forces involved in this step are van der Waals forces, electrostatic forces and hydrophobic interactions (Carpentier and Cerf, 1993; Gilbert et al., 1991; Van Loosdrecht et al., 1987). During this step bacteria can be easily removed by fluid shear forces, e.g. rinsing (Marshall et al., 1971). The next crucial step in the attachment process is the irreversible attachment of cells to the surface, described by Dunne (Dunne, 2002) as bacteria locking on to the surface by the production of exo-polysaccharides (EPSs) and or specific ligands, such as pili or fimbriae that may complex with the surface. At the end of this stage much stronger physical or chemical forces are required to remove the bacteria from the surface (e.g. scraping, scrubbing or chemical cleaners). In the transition from reversible attachment to irreversible attachment, various short range forces are involved, including covalent and hydrogen bonding as well as hydrophobic interactions (Kumar and Anand, 1998). Poortinga et al. (2001) expanded on the idea of covalent binding in bacterial attachment and suggested that bacteria either donated electrons to, or accepted electrons from the substratum (Jon et al., 2007). Many bacterial cells possess a capsule prior to attachment, the capsular and slime heteropolysaccharides contain uronic acids. The presence of uronic acid confers a net negative charge to the cell above pH 3 (Sutherland, 1980). In their study, Kennedy and Sutherland (1987) demonstrated that extracellular polymeric substances (EPSs) produced by marine bacteria may contain 20–50% of the polysaccharide as uronic acid which allowed them to have a negative charge. Recently, the discover of sulfate in polymers produced by prokaryotes other than Archaea and Cyanobacteria suggested that it allows EPS in seawater to have negative charge (Leppard et al., 1996). The overall negative charge gives the molecule a ‘‘sticky’’ quality and it is important to ensure the affinity of these EPSs for binding to cations such as dissolved metals (Brown and Lester, 1982), or eventually some mucus components having positive charge. Since surfaces and cells both tend to be anionic, the presence of positive ions in the mucus such as Ca2+ is important (Wolfaardt et al., 1999). Exopolymers including capsular polysaccharides and proteins are also important in bacterial adhesion to surfaces (Wolfaardt et al., 1999). The initial attachment can be reversible and is also related to the electrostatic interactions and cell wall hydrophobicity (Van Loosdrecht et al., 1990). Irreversible binding may occur as some bacteria, in close proximity to a surface, secrete large amounts of EPS slime (Costerton, 1984). Additional cross-linking of adjacent EPS chains enables permanent attachment to occur (Marshall, 1980). This process is influenced by electrolyte concentration (Fletcher, 1988). Bacteria may reversibly attach by secreting an exopolymer allowing them to stick to a surface and use surface associated nutrients (Hermannson and Marshall, 1985). This is followed by the secretion of a second polymer, which releases the attached bacterium (Mancuso Nichols et al., 2005). In marine sediments, many bacteria are enclosed within adherent biofilms which is a complex and heterogenic aggregation of microorganisms. This biofilm constitute a protected mode of growth that allows survival in a hostile environment. The structures that bacteria form in biofilm contain channels in which nutrients can circulate, and it has been demonstrated that cells in different regions of the biofilm exhibit different patterns of gene expression (Kolter and Losick, 1998; Whiteley et al., 2001). The bacteria in the biofilm are embedded in a matrix of EPSs produced by the individual cells (Flemming and Wingender, 2010; Costerton, 1999). This biofilm matrix is also commonly referred as glycocalyx; the extracellular substances are typically polymeric substances and commonly comprise a matrix of complex polysaccharides, proteinaceous substances and glycopeptides, lipids, lipopolysacharides and other materials that serve as a scaffold holding the biofilm together (Flemming and Wingender, 2010; Ma et al., 2006). More recent researchers discovered that as bacterial cell density within a biofilm increases, the bacteria may communicate with each other, in a cell-tocell signal (Dandekar et al., 2012). This can lead to the secretion of low molecular weight molecules that signal when the population has reached a critical threshold. This process, called quorum sensing, is responsible for the expression of virulence factors (Schauder and Bassler, 2001). For example, Pseudomonas aeruginosa produces some destructive proteases when the number of these bacteria reaches a high density in the biofilm. In this case, the infection is rarely resolved by the host defense mechanism (Fuqua et al., 1994; Corte´ s et al., 2011). In healthy fish, a cascade of complex components and reactions interferes to insure an equilibration between biofilm formation, biofilm components and biofilm control. According to Wingender et al. (1999), polysaccharides, proteins, nucleic acids, and lipids, are the major components of the matrix existing in the intracellular space of microbial biofilms and unattached aggregates in the marine environment. Nevertheless, Flemming et al. (1997) notice that, depending on their interaction with other organic (e.g. mucus, cells) or inorganic material (e.g. metals) in the marine environment, microbial exopolymers may be present in dissolved form or as biofilms and aggregates in a gel-like slime matrix. Three types of weak interactions provide cohesive forces in these gel-like matrices: dispersion forces, electrostatic interactions, and hydrogen bonding, which are significant when both, the frequency of the functional groups involved and the size of the polymers are considered (Flemming et al., 1997). EPSs enhance the survival of marine bacteria by influencing the physicochemical environment around the bacterial cell. Polysaccharides are released essentially during the stationary growth phase (Decho, 1990; Manca et al., 1996) and they represent the most abundant component ranging between 40% and 95% of the extracellular polymeric substances (Flemming and Wingender, 2001). Biochemical interactions between the bacteria and mucus, cells or tissues may be made possible by a hydrated matrix of exopolymers, capsular material including EPSs and others, which can provide a buffer against sudden changes in the adjacent osmotic environment (Dudman, 1977; Logan and Hunt, 1987; Decho, 1990). Such a stable environment may aid in the localization of secreted exoenzymes. These exoenzymes are essential in the adhesion mechanism, the sequestration of nutrients, the cellular uptake of small molecules and the cycling of both organic and inorganic material in the marine environment (Decho, 1990; Decho and Herndl, 1995). This explicate the affinity of bacteria in marine environment to fish mucus which is composed with hydrocarbon and may serves as carbon sources for bacteria. Moreover, the production of exopolymers enhances the growth and survival of microbes and the complex communities in which they are found (Wolfaardt et al., 1999). Extracellular polymers augment the ability of microbes to compete and survive in changing environmental conditions by altering the physical and biogeochemical microenvironment around the cell (Costerton, 1974). Consequently, these polymers provide protection (Bitton and Friehofer, 1978; Decho and Lopez, 1993) and ecosystem stability to the bacteria (Uhlinger and White, 1983; Dade et al., 1990; Mancuso Nichols et al., 2005). Through bibliographic data, several references reported an oriented bacterial adhesion to particles or surfaces. Several researchers have examined the role of flagella, which can cause oriented bacterial adhesion. Marshall et al. (1971), observed the preferential orientation, dependent upon motility of a flagellated Pseudomonas strain attaching to a glass surface. Also a Pseudomonas strain was observed by Fletcher to attach to hydrophilic surfaces via flagella, while assuming random orientations on hydrophobic surfaces (Fletcher, 1996). Experiments with the pathogens P. aeruginosa (Feldman et al., 1998) and A. hydrophila (Merino et al., 1997) have demonstrated the important role of flagella in establishing the initial interaction with mucosal surfaces or cells. Similarly, the effect of somatic antigen and flagella on the adhesion of V. alginolyticus was demonstrated by the effects of Oantiserum and H-antiserum on the bacterial adhesion. Somatic antigen and flagella played important roles in the V. alginolyticus adhesion (Chen et al., 2008). McClaine and Ford (2002a,b) found that flagellar rotation increases attachment rates of Escherichia coli bacteria to glass (McClaine and Ford, 2002a,b), although it could not be ascertained whether the bacteria were adhering by their cell bodies or by their flagella. Jones and coworkers (2003) demonstrated that three strains of E. coli having differing lengths of LPS surface molecules showed the same adhesion orientation, remaining at 90% end-on adhesion. This result seems to indicate that lipopolysaccharide (LPS) does not seem to be the important molecule involved in adhesion (Jones et al., 2003). Other bacterial substances are also implicated in the adhesion mechanism and in most cases; the adhesion has been reported to be mediated by proteins. Many studies reported that proteolytic enzymes, especially proteinase K, had a great effect on V. alginolyticus adhesion (Carnoy et al., 1994; Roos and Jonsson, 2002; Va`zquez-Jua´ rez et al., 2004). Because adhesion of V. alginolyticus to the mucus was reduced remarkably by proteinase K, cell-surface proteins of this bacterium may be important adhesins for adhesion. O-antigen LPS (known as the somatic antigen) was an important adhesin in A. hydrophila (Merino et al., 1996). Since adhesion can be a prerequisite for successful infection, adhesins can be considered primary virulence factors. Once adhesion occurs, the bacteria can induce the expression of other virulence genes and cause the activation of host cell signaling pathways (Finlay and Falkow, 1997; Toren et al., 1998). On the other hand, changes in pH and salinity over a wide range had little effect on the viscosity and stability of EPS produced by marine bacteria. Such results suggest that these EPSs may provide a buffer against shifting environmental conditions in the natural environment (Boyle and Reade, 1983). At present, it is known that many virulence genes of pathogenic bacteria are transcribed more efficiently at higher growth temperatures. In this regard, it was reported that induction of cascades of virulence factors occurs following P-pili mediated binding of E. coli to its host cell receptor (Zhang and Normark, 1996). In contrast, in their study on skin mucus of fish, Bordas et al. (1996) reported that the adhesive capability for skin mucus does not seem to be an essential virulence factor of pathogenic strains of Vibrio, since this specific interaction depended on several environmental factors, temperature and salinity being the most important. The absence of an inhibitory effect of mucus on the pathogenic microorganisms and the capability of the Vibrio strains to utilize mucus as a carbon source, could favor their settlement on the skin with a potential for infection of cultured stressed fish (Bordas et al., 1996). Using skin mucus as a carbon source and nutrients, attached bacteria can develop a biofilm which increase the opportunity to transfer gene between bacteria and may be significant for the transfer of resistance genes to associated susceptible bacteria. Gene transfer can convert a previously avirulent strain into a virulent pathogen (Montgomery and Kirchman, 1994). For these reasons, news and more virulent microbial phenotypes may be expressed when growing within a biofilm. Biofilms promote genetic diversity and maintain the high cell density needed for efficient genetic exchange. Several bacteria have plasmids allowing them to possess diverse characteristics. These plasmids can be transferred horizontally by conjugation to different species present in a biofilm (Nachamkin et al., 1993; Bordas et al., 1996). As it was previously indicated, virulence and pathogenicity of bacteria is often enhanced when growing as a biofilm, in this case, the immune responses are only directed toward those antigens on the outer surface of the biofilm because antibodies and other proteins or factor present in fish serum may fail to penetrate into the biofilm (Corte´ s et al., 2011). Furthermore, cells within the biofilm can modulate cytokine synthesis and remain hidden from antibody and complement factor recognition, and thus from subsequent white blood cell phagocytosis (Mccann et al., 2008). On the other hand, fish phagocytes are unable to effectively engulf a bacterium growing within a complex polysaccharide matrix, which may results in phagocytes releasing large amounts of pro-inflammatory enzymes and cytokines, leading to inflammation but also the destruction of nearby tissues (Sharp et al., 2006; Corte´ s et al., 2011). Possible suggestion of bacterial interaction with the components of mucus can be explained by the finding of some researchers who have observed polar (oriented) adhesion of bacteria due to specific mechanisms (Dawson and Ellen, 1990), such as adhesin-mediated adherence to fibronectin (Busscher and Weerkamp, 1987), lectinmediated adherence to Sepharose beads covalently derivatized with lactose (Loh et al., 1993), or pilus adherence to tracheal cells (Zoutman et al., 1991). Using a synthesized wettability gradient surface for non-flagellated bacteria, Ellen et al. (1998) have shown that Treponema denticola adheres flat onto hydrophobic surfaces but adheres on its ends to hydrophilic surfaces (Ellen et al., 1998). More recently, it was demonstrated that rod-shaped Klebsiella pneumoniae organisms adhere to surfaces in specific orientations by positioning them with a laser trap (Haruff et al., 2002; Jones et al., 2003). Nevertheless, in marine environment, the virulence of bacteria can be produced afterward a direct contact with fish skin or mucus, as well as, through some water soluble exoenzymes which are secreted and released by bacteria in the marine environment. Much more works are needed to understand the complex mechanisms involved in bacteria virulence.
4. Mucus responses to the adhesion of pathogens
Being in contact direct with their environment, fish need to develop effective strategies to overcome all types of challenges. Thus mucus is considered to be the first defense barrier of fish. Effectively, the fish skin mucus can neutralize or inhibit biofilm’s and bacterial effects since it can trap and immobilize pathogens before they can contact epithelial surfaces, because it is impermeant to most bacteria and many pathogens (Cone, 2009). This occurs because in this mucus layer, particles, bacteria, or viruses are entrapped and removed from the mucosa by the water current (Mayer, 2003). Attached to the mucus layer, bacteria of marine environment can resist and develop a biofilm. Nevertheless, to escape this challenge, healthy fish secrete and replace continuously their mucus layer which prevents the stable colonization of potential infectious microorganisms as well as invasion of metazoan parasites (Nagashima et al., 2003). Because of environmental conditions and fish’s health status, the mucus layer can be shed or digested; thus pathogens must move ‘‘upstream’’ through the unstirred layers of mucus adhering to the epithelial cells on the surface or penetrate a mucus ‘‘blanket’’ before it is shed (Cone, 2009). To prevent the pathogen adherence to the underlying tissues mucus has an underappreciated dynamic property to maintain an unstirred layer of mucus adjacent to epithelial surfaces despite vigorous shearing actions such as swallowing, coughing, intestinal peristalsis, and copulation (Cone, 2009; Esteban, 2012). Besides its role as biological barrier, mucus is involved in immune functions (Salinas et al., 2011) and its structure and function reflect the adaptation of the organism to the physical, chemical, and biological properties of the aquatic environment. In fact, mucus is composed essentially of mucins which are the most abundant components of the mucus; they are strongly adhesives, high molecular weight, filamentous, highly glycosylated glycoproteins (Esteban, 2012). Moreover, the mucus contain many other substances with biostatic and biocidal activity such as complement, C-reactive proteins, proteases (serine, cysteine, aspartic, and metalloproteases), lectins (galectin, pufflectin), lysozyme (acid and alkaline phosphatases), haemolysins, agglutinin, proteolytic enzymes, antimicrobial peptides (pardaxin, pleurocidins, parasin 1, hipposin, oncorhyncin III, oncorhyncin II, SAMPH1) and immunoglobulins (IgM and IgD, IgT/IgZ). These are present and have been identified in the fish epidermis and/or skin mucus. The mucus components and their role in immune defenses have been recently reviewed by Esteban (2012) and all of them could avoid the adhesion, colonization and/ or invasion of pathogenic bacteria. Many other factors could influence the bacterial adhesion to mucus. In fact, in their study, Chen et al. (2008) reported the effect of monosaccharides on bacterial adhesion. In fact, fructose exhibited a remarkable inhibition effect on the adhesion of V. alginolyticus to all mucus investigated while mannose and galactose significantly inhibited the bacterial adhesion to gill mucus. Mannose also inhibited the bacterial adhesion to hindgut mucus but glucose showed no significant inhibition effect on the bacterial adhesion to any mucus. Moreover, the data obtained from the characteristics of mucus show thatthere is more IgM and lysozyme in skin mucus than in mucus from other fish parts; this might be one of the reasons why the adhesion of V. alginolyticus to skin mucus is less than to other mucus. It has been reported that the gastrointestinal tract is the portal of entry for V. anguillarum into fish (Horne and Baxendale, 1983; Oisson et al., 1996) while the intestinal tract rather than the gill or skin was also the entry portal of V. alginolyticus into large yellow croaker (Chen et al., 2008). Anyway, it remains slightly unclear whether these components of mucus have a bactericidal effects, thus investigations should be developed to better understand the bactericidal effect of these components of mucus. This can be useful to develop a biological efficient treatment of fishes cultured in farms. Regardless of the composition of the mucus, it seems that the infection of fish with pathogenic bacteria depends strongly on the bacterial and fish species. Effectively, the results found by Magarinos et al. (1995) suggested collectively that a possible portal of entry for Tenacibaculum maritimum (Flexibacter maritimus) into the fish body can be the skin. However, with Photobacterium damselae subsp. piscicida (Pasteurella piscicida) the pathways of entry may vary depending on the host. Some fish can be infected by ingestion of the pathogen. Unrelated to the route of entry, upon reaching to invades the fish, the success of the pathogen to establish an infection or causing a disease will depend on other factors, including its ability to neutralize or evade the immune system of the fish and its ability to scavenge the nutrients required for its growth. In their study, using two pathogen bacteria (P. piscicida and F. maritimus), Magarinos et al. (1995) demonstrated that a strong bacterial adhesion to an organ or tissue does not necessarily result in the occurrence of disease. Besides, the infection depends strongly on the mucus composition which depends on the fish status and environmental conditions. In their study Chen et al. (2008) reported that the temperature and proteolytic enzymes influences bacterial adhesion since they can change the mucus composition. In fact, adhesion was shown to be mediated by lectins presented on the surface of the infectious organism that bind to complementary carbohydrates on the surface of the host tissues (Acord et al., 2005). Soluble carbohydrates, recognized by the surface lectins of bacteria, block the adhesion of bacteria to animal cells in vitro (Sharon, 2006). Thus, a phenomenon of specificity or affinity of some bacteria regarding to specific type of mucus (gill mucus, skin mucus or intestinal mucus) should be studied and could be useful to develop suitable strategies of prevention in fish farms. In their experiment realized in vitro, Chen et al. (2008) demonstrated also that incubating of the mucus at 50 8C for 10 min induce more bacterial adherence to skin mucus compared with the control group, whereas incubating at 75 8C or 100 8C for 10 min reduced the number of V. alginolyticus adhering to gill mucus. This can be explicated by the destruction of binding sites of V. alginolyticus in mucus. However, no significant difference was found in the bacterial adhesion to intestinal mucus after heating which support the difference in composition of mucus according to their origin, as it has been previously mentioned. Considering that the majority of bacteria found in the marine environment are psychrophilic to mesophilic (growth temperature between 4 and 30 8C) (Cho and Giovannoni, 2004), it seems to be normal that these bacterial populations lost the adhesion propriety at high temperature. In contrast, the matrix of mucus, due to its heterogeneity can be thermotolerent. Nevertheless, more detailed studies are needed to understand chemical variation of mucus under temperature higher than 50 8C. Eventual interesting characteristics of heated mucus can be useful to prevent fish diseases. Knowledge of the variation of chemical component of mucus course temperature, in relation with adhesive characteristics of mesophilic and thermophilic pathogen bacteria should be very useful for designing more effective prophylactic strategies.
5. Importance of fish microbiota
External mucus secretion cannot be studied independently to the general health status of fish. Indeed, fish health is strongly dependent on the internal microbiota which helps in nutrients uptake and protect fish from pathogenic invasion (Ringo et al., 2000; Makridis et al., 2005). Adhesion capacity and growth on or in intestinal or external mucus has been demonstrated in vitro for fish pathogens like V. anguillarum and A. hydrophila (Garcia et al., 1997; Krovacek et al., 1987) and for candidate probiotics such as Carnobacterium strain K1 (Jo¨born et al., 1997) and several isolates inhibitory to V. anguillarum (Olsson et al., 1992). In one of these studies, the aim was to measure the in vitro capacity of the strains to adhere to and grow in turbot intestinal mucus in order to investigate their potential to colonize farmed turbot as a means of protecting the host from infection by V. anguillarum (Olsson et al., 1992). The intestinal isolates generally adhered much better to a film of turbot intestinal mucus, skin mucus, and bovine serum albumin than did V. anguillarum, indicating that they could compete effectively with the pathogen for adhesion sites on the mucosal intestinal surface (Verschuere et al., 2000). Mechanisms that may be implicated include the production of antimicrobial substances such as organic acids or bacteriocins, competition for nutrients or adhesion receptors, inhibition of virulence gene expression, and enhancement of the immune response (Balcazar et al., 2007). On the other hand, intake of probiotics has been demonstrated to ameliorate fish’s resistance to pathogen (Tapia-Paniagua et al., 2012) and 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. Uses of probiotics may provide protection by competition for host extracellular matrix-binding sites, thereby blocking adhesion and spread of the pathogens; stimulation of host-cell immune defenses; or triggering cell-signaling events that deactivate the production of virulence factors and the subsequent onset of sepsis (Reid et al., 2001). The modes of action of probiotics are as follows: production of inhibitory compounds; competition for chemicals or available energy; competition for adhesion sites; improvement of water quality; source of macro- and micronutrients; and enzymatic contribution to digestion. It was suggested that the inhibitory effects of lactic acid bacteria (LAB) may be due to either individual or joint production of organic acids, bacteriocins or hydrogen peroxide (Klaenhammer, 1993; Vandenbergh, 1993). Although bacteriocins of LAB were tested to be commonly active against Gram-positive spoilage and pathogenic bacteria, these bacteriocins exhibit limited or no antimicrobial action against Gram-negatives (Abee et al., 1995). However, a nisin-like bacteriocin produced by Lc. lactis subsp. lactis A 164, isolated from kimchi (Korean traditional fermented vegetables), was active against closely related LAB and Salmonella typhimurium (Choi et al., 2000). Chung and Yousef (2005) reported that Lb. curvatus OSY-HJC6 produces a bacteriocin-like agent active against Gram-negative strains such as Escherichia coli p220, E. coli O157:H7, Salmonella enteritidis, S. typhimurium, and P. fluorescens (Balcazar et al., 2007).
Even if probiotic bacteria, known to enhance immune system of fish (Esteban, 2012), the orientation is to use the probiotics allowing high capacity of adhesion. However for the pathogen bacteria it seems that a natural selection is operating, and the most adhesive and resistant bacteria have the opportunity to survive and to induce disease. It has been suggested that the presence of large and diverse indigenous populations of bacteria renders the gastrointestinal tract relatively resistant to invasion by non-indigenous species. Some bacterial isolates from the turbot gastrointestinal tract appear to secrete substances that inhibit the growth of V. anguillarum (Westerdahl et al., 1991). Further, it appears that populations of bacteria in the intestinal tract are controlled by substrate competition (Freter et al., 1983; Garcia et al., 1997), competition for nutrients or adhesion receptors (Nikoskelainen et al., 2001; Vine et al., 2004; Balcazar et al., 2007), inhibition of virulence gene expression, and enhancement of the immune response (Nikoskelainen et al., 2003; Kim and Austin, 2006; Balcazar et al., 2007; Gomez and Balcazar, 2008). Burghoff and coworkers have demonstrated thatthe ability of both E. coli and S. typhimurium to colonize the mouse colon is dependent upon the ability of each organism to express proteins that allow the utilization of colonic mucus components (Burghoff et al., 1993; Garcia et al., 1997). Effectively, the phenomenon of competition and growth inhibition by spent culture liquid has been well proved. In their study, Balcazar et al. (2007), demonstrated that the adhesion of F. psychrophilum and R. salmoninarum were not inhibited by LAB strains (used as probiotics), whereas, the fish pathogenic strains A. salmonicida subsp. Salmonicida and Y. rukeri were inhibited by all used LAB strains. Additionally, the adhesion of C. piscicola was significantly reduced between by strains Lc. lactis subsp. lactis CLFP 100, Lc. lactis subsp. cremoris CLFP 102, Lb. curvatus CLFP 150, Lb. sakei CLFP 202 and Leuc. mesenteroides CLFP 196. In another study (Tuomola et al., 1999),two strongly adhesive strains of lactobacilli significantly increased in vitro adhesion of S. typhimurium to human intestinal mucus. This clearly demonstrates that each probiotic strain should be judged on its own merits and that extrapolation from related strains is not acceptable, as suggested by previous studies (Ouwehand et al., 1999; Balcazar et al., 2007).
6. Inhibitory compounds present in fish mucus
Depending on several factors, such as the fish species, health, environment, mucus origin and microbiota colonizing the mucus, many types of interaction could be implicated to prevent pathogen infection. It is important to take into account that, additionally to the immune system, fish have a bacterial community that can inhibit the adhesion or the development of pathogens. Effectively, some bacteria produce inhibitory compounds, particularly in the digestive tract, and may be responsible for controlling the colonization of potential pathogens in fish (Ringo et al., 2000; Makridis et al., 2005). For example a Vibrio sp. recovered from the intestine of a spotnape ponyfish (Leiognathus nuchalis) in Japanese coastal waters inhibited the causal agent of pasteurellosis/pseudotuberculosis, i.e. P. damselae subsp. piscicida. Specifically, the inhibitory compound was heat-labile and proteinaceous, with a molecular mass of <5 kDa, and was considered to be possibly a bacteriocin or a bacteriocin-like substance (Sugita et al., 1997). Similarly, some bacteria, isolated from the digestive tract of halibut (Hippoglossus hippoglossus) larvae, were found to be capable of inhibiting growth of pathogenic Vibrio sp. (Bergh, 1995). All these bacterial strains were Gram-negative rods, most of which were fermentative and produced catalase and oxidase (Austin, 2006). The skin epithelium and other mucosal surfaces of fish are the sites in which anti-microbial peptides are particularly likely to be abundant. In many other animal groups the surface mucosa produces a battery of nonspecific microbicidal proteins to prevent harmful systemic infections that would result if microbes breached this barrier (Bevins, 1994; Simmaco et al., 1998). It is well established that skin secretions of fish contain a number of innate humoral defense factors, including complement factors (Yano, 1996), lectins (Buchmann, 2001), proteases (Hjelmeland et al., 1983) and lysozymes (Hikima et al., 1997; Smith et al., 2000). More recent investigations have started to reveal that the surface mucosa of some fish also contains a range of anti-microbial peptides (Richards et al., 2001). Jorge et al. (2002), demonstrated that histone H2A purified from trout (O. mykiss) mucus was found to be a remarkably potent anti-bacterial agent. Its minimal inhibitory concentrations are in the submicromolar range and approx. 10 times lower (on a molar basis) than those of cecropin against sensitive bacteria. The study revealed that there is a direct proportionality between anti-bacterial activity, protein concentration and incubation time, in common with other anti-microbial peptides (Cole et al., 1997). Furthermore, its anti-bacterial action is exerted rapidly, i.e. 30 min incubation at 0.3 lM is sufficient to totally inactivate the Gram-positive test bacteria. Importantly, at this concentration the protein is not lytic for trout erythrocytes, although it has haemolytic effects at higher concentrations (Jorge et al., 2002). Another study was realized on 940 aerobic and anaerobic bacteria isolated from digestive tracts of fish, water and sediments, and it demonstrates that some of the isolates (i.e. Bacteroides type A and other Bacteroidaceae representatives) inhibited the growth of some pathogenic bacteria including A. hydrophila, A. salmonicida, E. coli, and Staphylococcus aureus (Austin, 2006). In another research carried out by the same group, it was reported that, 2.7% of isolates from intestinal bacteria derived from seven coastal fish in Japan, inhibited the human and eel pathogen V. vulnificus (Sugita et al., 1998). Thus, marked inhibition was displayed by 15 isolates, comprising eleven Vibrionaceae representatives, three coryneforms, and one Bacillus strain NM 12; the latter demonstrated the most profound antimicrobial activity. This revealed that one of the inhibitory compounds, which was determined to be a heat labile siderophore of <5 kDa molecular weight, inhibited the growth of 227 out of 363 (62.5% of the total) of the intestinal bacterial cultures (Sugita et al., 1998). Others studies have achieved this level of success. For example, of >400 bacteria recovered from turbot, 89 inhibited the growth of the fish pathogen V. anguillarum (Olsson et al., 1992). Similarly, of >400 isolates from the intestine and the external surface of farmed turbot, 28% (mostly from the digestive tract) inhibited A. salmonicida, A. hydrophila, and V. anguillarum (Westerdahl et al., 1991).