PROGRAMA DE EDUCACIÓN EMOCIONAL EN LA ESCUELA La escuela es uno de los medios más importantes a través del cual el niño aprenderá y se

Like other organisms, fish are constantly interacting with their environment, which may potentially contain pathogenic microorganisms. When microbes invade the host, cellular and humoral innate defence mechanisms including cytokines, the antioxidant defences, acute phase proteins or the cellular responses are activated (Kiron, 2012). The teleost immune system may be categorized into two types, these being ‘innate’ and ‘acquired’, as presented in Figure 1.1.

The innate immune system is further divided into two parts - cellular and humoral immunity. Antimicrobial peptides, complement components, lysozyme, pentraxins, transferrin, antiproteases, lectins and natural antibodies constitute the innate humoral effectors, whereas the innate cellular immune parameters are mediated by nonspecific cytotoxic cells and phagocytes (monocytes/ macrophages and neutrophils) (Magnadóttir, 2006; Gómez and Balcázar, 2008). Fish have a low specificity immune system with a shorter response, a limited antibody production (IgT and IgM) and a weak memory (Trichet, 2010).

The innate immune response is activated through target molecules including lipopolysaccharides (LPS), peptidoglycans, bacterial DNA and double- stranded viral RNA that are so-called pathogen-associated molecular patterns (PAMP) to microbes, which induce the immune response against infections of fish (Ringø et al., 2012).

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Figure 1.1 Schematic representation of immune response of fish following an

encounter with a pathogen. Adapted from Shoemaker et al. (2001).

Mucus, which is secreted by goblet cells in the epithelial barriers including skin and the gut, plays a crucial role in preventing pathogenic microorganism from reaching the tissues due to several components that it contains: immunoglobulin, lysozyme, transferrin, complement system, antimicrobial peptides etc. (Trichet, 2010; Lazado and Caipang, 2014).

Adaptive (or acquired) immunity is comprised of two parts - humoral and cellular immunity. Immunoglobulins, which are produced by B-cells constitute the adaptive humoral effectors, whereas the cellular adaptive immune parameters are mediated by T-lymphocytes and B-cells (Denev et al., 2009). Magnadóttir (2006) reported that a variety of external and internal factors, including temperature fluctuation, handling and crowding stress may negatively affect the activity of the innate immune system, whereas prebiotics, probiotics and immunostimulants can improve its activity. Additionally,

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Shoemaker et al. (2001) reported that innate and acquired immune profiles may be influenced by factors such as the size, age and weight of the fish themselves.

Lysozyme is a component of the humoral innate immune system. It is a cationic enzyme with the ability to destroy the cell walls of many Gram- positive bacteria by breaking the glycosidic bonds between N-acetylmuramic acid and N-acetylglucose-amine in the peptidoglycan cell wall. It is also active against fungal cell walls and some strains of Gram-negative bacteria (Ellis, 2001; Villa and Crespo, 2010). Although enzymatic damage of peptidoglycan represents the main mode of antibacterial activity of lysozyme, it is well accepted that bacteria could be killed by non-enzymatic techniques (Villa and Crespo, 2010). This non-enzymatic activity can be categorized into two types. Firstly, in the lytic mode, as lysozyme is cationic, it can activate bacterial autolytic enzymes (autolysins). Secondly, in the non-lytic mode, the cell is killed via disruption of the peptidoglycan portion of the membrane, ultimately leading to cell lysis (Fischetti, 2008). In fact, it is possible that non- enzymatic activity destroys more bacteria compared with the enzymatic split of peptidoglycan (Masschalck and Michiels, 2003).

In general, only Gram-positive bacteria are killed by lysozyme, whereas Gram-negative bacteria are generally protected against lysozyme’s enzymatic activity by their external membranes (Villa and Crespo, 2010). However, lysozyme has some developmental modifications, which increase its ability to kill Gram-negative bacteria (Ibrahim et al., 2002), by facilitating the movement of molecules through the outer membrane (Masschalck and

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Michiels, 2003). It is considered that the physiology of fish, environmental condition, infections and levels of stressors all affect the activity of lysozyme (Saurabh and Sahoo, 2008).

Cellular components of the innate immune system such as neutrophils, monocytes and macrophages play an important role in the defence against bacteria, viruses, and parasites in fish and shellfish (Harikrishnan et al., 2011). Phagocytosis activity has been reported to be the second line of immunity and that phagocytic cells will proliferate quickly to deactivate pathogenic microorganisms (Trichet, 2010).

Granulocytes, monocytes/macrophages and natural cytotoxic cells constitute the cellular part of the innate immune system in fish. Natural cytotoxic cells are responsible for fighting viral infections; their receptors distinguish proteins presented at the surface of viral particles (Trichet, 2010). Granulocytes are classified into three distinctive types, depending on their morphology: neutrophils, eosinophils and basophils. Neutrophils exist in the circulation in low numbers, but under activation conditions as a result of presence of cytokines, specific activation molecules or other bacterial components, neutrophils migrate toward the source of inflammation where the infection occurred whether in the circulation system or in the tissues (Suzuki and Iida, 1992). Eosinophils are ordinarily less numerous than neutrophils, but the former can be appear in greater levels as a result of presence of parasitic infections (Beutler, 2004; Alvarez-Pellitero, 2008). It has been reported that phagocytotic cells, including macrophages and neutrophils are able to engulf microbes and kill them using enzymes and reactive oxygen species (ROS),

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especially, hydroxyl radicals, singlet oxygen, oxygen halides and hydrogen peroxide during the respiratory burst activity (Ellis, 2001). On the other hand, two types of cells are responsible for the function of the adaptive immune system: the B-lymphocytes and the T-lymphocytes, these are considered the third line of immune system (Trichet, 2010). B-lymphocytes are designed to produce a specific antibody on their cell surface membrane while antigens detected by macrophages stimulate T-lymphocytes, thus they provoke B- lymphocytes to produce specific antibodies against the determined antigen.

Cytokines are proteins, which are related to both the specific and non- specific immune response. Immune cells in particular produce numerous cytokines, including groups of interleukin, interferon, tumor necrosis factor, transforming growth factor-β and various chemokines (Gómez and Balcázar, 2008). Cytokines and chemokines are secreted by leucocytes (in particular neutrophils) in response to microbial antigens or substances released from injured cells, and act as intracellular signals to regulate the components of the innate and adaptive immune response (Magnadóttir, 2006). Recently, many investigators have been able to use the mRNA expression of cytokine genes in response to probiotic bacteria as a technique for measuring immune responses (Kim and Austin, 2006a; Panigrahi et al., 2007; Mansfield et al., 2010; Pérez-Sánchez et al., 2011b). The gut associated lymphoid tissue (GALT), acts as a barrier to the entry of pathogens and contains leucocyte populations which are exposed to the external environment. GALT in teleost fish lacks specialized structures such as the Peyer’s patches of mammals but the gut contains populations of leucocytes, including macrophages,

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lymphocytes, mast cells, granulocytes and plasma cells (Pérez et al., 2010). The immune cells are intensively present in the posterior intestine of fish, which act to be an important antigen presenting cells (APCs); IgT, a specialised mucosal antibody which is an isotype of mammalian IgA, is also present in the gut mucus of fish(Rauta et al., 2012). Commensal bacteria are also present in the GALT, which are implicated in the induction of local immune responses (Pérez et al., 2010). Figure 1.2 represents the structure of the gut mucosa surface with different profiles of both innate and adaptive immune systems.

Figure 1.2 Representation of intestinal mucosal surfaces in the teleost fish. The

cellular components of the innate immune system (dendritic cells, macrophages, granulocytes and mast cells, the location of B and T cells and the secretory components (SC) of the polymeric immunoglobulin receptor (pIgR) are displayed. Commensal bacteria and antimicrobial peptides (AMPs) are shown in the outer mucosal surface. Adapted from Gómez et al. (2013).

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