Volume 39, Issue 3 pp. 223-239

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Lysozyme: an important defence molecule of fish innate immune system

Shailesh Saurabh,

Shailesh Saurabh

Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, India

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P K Sahoo,

P K Sahoo

Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, India

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Shailesh Saurabh,

Shailesh Saurabh

Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, India

Search for more papers by this author

P K Sahoo,

P K Sahoo

Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, India

Search for more papers by this author

First published: 24 January 2008

https://doi.org/10.1111/j.1365-2109.2007.01883.x

Citations: 1,343

Correspondence: P K Sahoo, Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751 002, India. E-mail: [email protected]

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Abstract

The innate immune system of fish is considered to be the first line of defence against a broad spectrum of pathogens and is more important for fish as compared with mammals. Lysozyme level or activity is an important index of innate immunity of fish and is ubiquitous in its distribution among living organisms. It is well documented that fish lysozyme possess lytic activity against both Gram-positive bacteria and Gram-negative bacteria. It is also known to be opsonic in nature and activates the complement system and phagocytes. It is present in mucus, lymphoid tissue, plasma and other body fluids of freshwater and marine fish. It is also expressed in a wide variety of tissues. Lysozyme activity has been shown to vary depending on the sex, age and size, season, water temperature, pH, toxicants, infections and degree of stressors. Here, we review our current understanding of different types of lysozyme and their expression and its role in fish innate immune system.

Introduction

Immunity is an important physiological mechanism in animals for protection against infection and preservation of internal homoeostasis. The immune system is usually divided into two parts: innate immunity that recognizes invading microbes by germline-encoded molecules and adaptive immunity where recognition depends on molecules generated by somatic mechanisms during the ontogeny of each individual organism (Medzhitov & Janeway 1997). The innate immune system is phylogenetically older and is found in some form in all multicellular organisms, whereas the adaptive system appeared about 450 million years ago and is found in all vertebrates, except jawless fish (Zarkadis, Mastellos & Lambris 2001). The immune system protects the organisms from attack by a broad spectrum of invading microorganisms. It uses specialized organs designed to filter out and respond to microbes entering the body's tissues and a mobile force of molecules and cells in the blood stream to respond rapidly to attack. The system can fail, giving rise to immunodeficiency, or may over react against foreign microbes, giving rise to tissue damage. It has complex and sophisticated mechanisms to regulate it (Lydyard, Whelan & Fanger 2000).

The fish, because of its earliest evolution, mostly depends on non-specific/innate/natural immunity and also its immune system is less well developed as well as studied. When a fish comes in contact with foreign agents/pathogens, it is the innate or natural immunity/defence system that protects or fights to get rid of the pathogens/agents (Sahoo 2004, 2006). Innate immunity essentially serves as the host's first line of defence against invasion of pathogens whereas adaptive/acquired/specific immunity plays a vital role in protection against recurrent infections by generating memory cells (cell-mediated immunity) and specific soluble and membrane-bound receptors (humoral immunity) such as T-cell receptors and immunoglobulin (Ig) that allow for the fast and efficient elimination of the specific fish pathogens (Ellis 2001; Swain 2006). In both types of immunity, cells and molecules play an important role. Furthermore, acquired immunological mechanisms are highly specific, complex and are marked by diversity and memory. However, the acquired or specific immune response usually takes days to develop (Magnadottir 2006) and because it targets pathogen-specific features, it only occurs after the pathogen is present for a certain amount of time in one form or the other (Dixon & Stet 2001). The recognition of pathogen-specific molecules or features, however, allows the acquired immune response to increase in magnitude and speed with every subsequent exposure to the same pathogen. The presence of an acquired immune system, however, has not made innate immunity obsolete (Galindo-Villegas & Hosokawa 2004). The innate immune system plays an instructive role in the acquired immune response and homoeostasis and is therefore equally important in higher vertebrates. Moreover, by functioning as a first line in host defence, innate immune responses can avert many microbial attacks or keep them in check until an efficient acquired immune response has been developed. The most important feature of this innate immune system is that it exists before or just after hatching whereas the specific immunity takes a few weeks to months to develop during ontogeny. The important molecules viz., lysozyme, myeloperoxidases, superoxides, acute-phase proteins, interferon, complement, properdin, lysins and agglutinins, are some of the important innate immune parameters and have often been used as indicators of aquatic stress response and disease resistance.

Lysozyme and its function

Lysozyme (muramidase, EC 3.2.1.17) is an important defence molecule of the innate immune system, which is important in mediating protection against microbial invasion. It is a mucolytic enzyme of leucocytic origin. Lysozyme is widely distributed in bacteriophages, microbes, plants, invertebrates and vertebrates (Jolles & Jolles 1984) and is also found in a large variety of animal secretions such as mucus and saliva, in many tissues including blood and in the cell vacuoles of plants. It splits the β (1→4) linkages between N-acetylmuramic acid and N-acetylglucosamine in the cell walls (peptidoglycan layers) of Gram-positive bacteria, thus preventing them from invading. Gram-negative bacteria are not directly damaged by lysozyme. When the outer cell wall of Gram-negative bacteria is disrupted due to the action of complement and other enzymes exposing the inner peptidoglycan layer of bacteria, then lysozyme becomes effective. Besides an antibacterial function, it promotes phagocytosis by directly activating polymorphonuclear leucocytes and macrophages or indirectly by an opsoninic effect. The enzyme also attacks structures containing muramic acid, hydrolyses glycol chitin and has a restricted degrading effect on chitin (a linear polymer of N-acetylglucosamine), which is a major component of the cell walls of fungi and the exoskeletons of certain invertebrates. Furthermore, lysozyme is a food-safe, antimicrobial enzyme that can also produce gels and films. Recently, Bower, Avena-Bustillos, Olsen, McHugh and Bechtel (2006) reported that fish-skin gelatin gels and films, when formulated with lysozyme, may provide a unique, functional barrier to increase the shelf-life of food products.

Lysozymes are synthesized in both the liver and the extra-hepatic sites; however, the kinetics of synthesis and secretion are not yet clear (Bayne & Gerwick 2001). High activities of the enzymes are found against Micrococcus lysodeikticus in the lymphomyeloid tissues of elasmobranchs and in the plasma of teleosts. However, lower activities are demonstrated in the spleen, and little or no activity is observed in the Atlantic hagfish and cod. Like mammals, the lysozyme in fish occurs mainly in neutrophils, monocytes and a small amount in macrophages. It is reported that the kidneys have highest lysozyme levels, followed in descending order by the alimentary tract, spleen, skin mucus, serum, gills, liver and muscle (Lie, Evensen, Sorensen & Froysadal 1989). The close association of lysozyme with cells of the immune system may indicate that the enzyme contributes to defence against infectious diseases. The enzyme is a single polypeptide chain of about 120 amino acids. It has an isoelectric point between pH 9 and 11, its molecular weight is about 14.4 kDa and its optimal activity occurs between pH 5 and 7 (Alexander & Ingram 1992). The lysozyme of freshwater fish shows one pH optimum at 5.4 and requires a low buffering concentration for its activity while marine fish lysozyme has two optimum pH at 6.2 and 9.2 in relatively high buffer molarity (Sankaran & Gurnani 1972). Lysozymes are measured using three methods namely: turbidimetric assay (Parry, Chandau & Shahani 1965), lysoplate assay (Ossermann & Lawlor 1966) and lysorocket electrophoresis (Virella 1977).

In higher vertebrates, lysozymes are involved in a broad battery of defence mechanisms, embracing actions such as bacteriolysis, opsonization, immune response potentiation as well as restricted antiviral and antineoplastic activity (Jolles & Jolles 1984). In addition to their role in defence, lysozyme has digestive tasks (Dobson, Prager & Wilson 1984). In fish, lysozyme, an enzyme with antibiotic properties that is released by leucocytes, has a broader activity than mammalian lysozyme (Demers & Bayne 1997) and has been frequently used as an indicator of non-specific immune functions, which is of primary importance in combating infections in fish. Fish skin mucus contains many humoral non-specific defence factors for example, lysozyme, complement, interferon, C-reactive protein, lectin (haemagglutinin), haemolysin and transferrin. These innate immunity molecules are especially important for fish species, because fish live in a water medium rich in pathogens (Ingram 1980) and thus play a key role in maintaining homoeostasis in the animal. Fish lysozyme is mainly distributed in the head kidney, which is rich in leucocytes, and at sites such as the gill, skin, gastrointestinal tract and eggs, where the risk of bacterial invasion is very high (Table 1), which emphasizes the role of lysozyme in defence, especially in the first line of defence mechanisms (Murray & Fletcher 1976; Lie et al. 1989). Lysozyme activity has been identified in monocytes, and neutrophils of the plaice, Pleuronectes platessa L., probably contribute to serum lysozyme activity because their number increases concomitantly with serum lysozyme levels (Fletcher & White 1973).

Table 1. Lysozyme activity detected in teleosts tissues

Common name	Scientific name	Organs	Reference(s)
Tilapia	Tilapia mossambica	Gill, serum, liver	Sankaran and Gurnani (1972)
Scat	Scatophagus argus	Gill, Liver	Sankaran and Gurnani (1972)
Perch	Therapon puta	Gill, Liver	Sankaran and Gurnani (1972)
Haddock	Melanogrammus aeglefinus	Pseudobranch, kidney	Fange, Lundblad and Lind (1976), Lie et al. (1989)
Pollack	Pollachius pollachius	Pronephros, pseudobranch, red blood cells, spleen	Fange et al. (1976)
Herring	Clupea harengus	Plasma, red blood cells	Fange et al. (1976)
Plaice	Pleuronectes platessa	Neutrophils, peritoneal macrophages, gill cartilage and epithelium, spleen, kidney, skin and mucus	Murray and Fletcher (1976)
Bonito	Katsuwonus pelamis	Pyloric cecum	Mochizuki and Matsumiya (1981)
Yellowtail	Seriola quinqueradiata	Pyloric cecum, stomach, intestine, liver, skin mucus, kidney, serum	Mochizuki and Matsumiya (1981), Takahashi, Kajiwaki, Itami and Okamoto (1987), Kusuda, Kawahara and Hamaguchi (1987)
Rainbow trout	Salmo gairdneri	Skin mucus, gut mucus, kidney, gill, liver, muscle, alimentary tract, spleen	Hjelmeland, Christie and Raa (1983), Grinde, Jolles and Jolles (1988), Lie et al. (1989), Kim and Austin (2006)
Common carp	Cyprinus carpio	Skin mucus	Takahashi, Itami and Konegawa (1986)
Ayu	Plecoglossus altivelis	Skin mucus, kidney, intestine, gill mucus	Itami, Takahashi and Kawahara (1986)
Eel	Anguilla japonica	Skin mucus, kidney, serum, intestinal mucus	Kawahara and Kusuda (1988), Nielsen and Esteve-Gassent (2006)
Atlantic salmon	Salmo salar	Kidney, intestine	Lie et al. (1989), Sveinbjornsson, Olsen and Paulsen (1996)
Sea char	Salvelinus alpinus	Kidney	Lie et al. (1989)
Turbot	Scopthalmus maximus	Kidney	Lie et al. (1989)
Blue gourami	Trichogaster trichopterus	Plasma, kidney	Low and Sin (1998)
Japanese char	Salvelinus leucomaenis	Plasma	Miyazaki (1998)
Japanese flounder	Paralichthys olivaceus	Plasma, Recombinant protein (cDNA from kidney)	Miyazaki (1998), Minagawa et al. (2001), Hikima et al. (2001)
Asian catfish	Clarias batrachus	Serum	Kumari and Sahoo (2005a, b)
Rohu	Labeo rohita	Serum	Sahoo, Kumari and Mishra (2005)
Catla	Catla catla	Serum	Sahoo et al. (2005)
Mrigal	Cirrhinus mrigala	Serum	Sahoo et al. (2005)
Atlantic cod	Gadus morhua	Intestine	King, Buckley and Berlinsky (2006)
Nile tilapia	Oreochromis niloticus	Plasma, liver, skin, mucus	Taoka et al. (2006), Welker, Lim, Yildirim-Aksoy and Klesius (2007)
Great sturgeon	Huso huso	Liver, kidney, spleen, serum	Khoshbavar-Rostami, Soltani and Hassan (2007)
Japanese eel	Anguilla japonica	Serum, mucus	Ren, Koshio, Ishikawa, Yokoyama, Micheal, Uyan and Tung (2007)

Day, Marceau-Day and Ingram (1978) reported that lysozyme attacks the lipopolysaccharide layer of Gram-negative fish pathogen Pseudomonas aeruginosa and other cell structures like the cytoplasmic membrane (Iacono, MacKay, DiRienzo & Pollock 1980), which causes damage to the outer cell membrane, thus allowing additional lysozymes to reach and injure deeper-lying structures like the cytoplasmic membrane. The latter may thus be rendered so permeable as to result in the loss of cell viability without lysis. The two lysozyme variants (types I and II), purified from the kidney of rainbow trout, show potent antibacterial activity of the type I variant against seven Gram-negative bacterial species in comparison with standard hen egg white lysozyme having antibacterial activity against some non-pathogenic bacteria (Grinde 1989). The fish lysozyme has substantial antibacterial activity over mammalian lysozymes not only against Gram-positive bacteria but also against Gram-negative bacteria in the absence of complement. Two different types of lysozyme isolated from the skin mucus of Ayu fish (Plecoglossus altivelis) possessed high bacteriolytic activity against formalin-killed bacterial cells of Aeromonas hydrophila and Pasteurella piscicida, but low activity against those of Vibrio anguillarum, which is a pathogenic agent of Ayu fish (Itami, Takehara, Nagano, Suetsuna, Mitsutani, Takesue & Takahashi 1992). The lysozyme obtained from coho salmon eggs is bactericidal to A. hydrophila, Aeromonas salmonicida and Carnobacterium piscicola at a concentration of 700 μg mL⁻¹ but not to Renibacterium salmoninarum. These findings indicate that lysozyme plays a role in preventing the mother-to-progeny (vertical) transmission of some bacterial fish pathogens, and its failure to kill R. salmoninarum helps to explain why this organism is readily transmitted vertically (Yousif, Albright & Evelyn 1994). Nakamura, Gohya, Losso, Nakai and Kato (1996) demonstrated the therapeutic effect of lysozyme–galactomannan or lysozyme–palmitic acid conjugates against Edwardsiella tarda infection in carp.

In nature, lysozyme is found as a monomer. The lysozyme dimer is significantly less toxic than its monomer, and its biological activity has been ascertained in cases of both viral and bacterial infections. Siwicki, Klein, Morand, Kiczka & Studnicka (1998), Siwicki, Morand, Klein & Kiczka (1998) reported that dimerized lysozyme (KLP-602) stimulated the cellular and humoral defence mechanisms of fish and provided protection against furunculosis and infectious pancreatic necrosis virus (IPNV). Rymuszka, Studnicka, Siwicki, Sieroslawska and Bownik (2005) reported that the dimer of lysozyme (KLP 602) acts as an immunomodulator and useful for stimulation of cellular and humoral immunity after experimentally induced suppression of carp by pesticides.

Patrzykat, Zhang, Mendoza, Iwama and Hancock (2001) reported that activities of Coho salmon skin extracts containing lysozyme and hen egg white lysozyme against V. anguillarum were potentiated by histone-derived peptides, which had no activity on their own. The interesting aspect of this finding with respect to lysozyme is that it suggests that the potentiation of lysozyme activity by the histone peptide could result from a transient or permanent peptide-induced break in the outer membrane of the Gram-negative species, allowing lysozyme to react to the cell wall and destroy the bacteria. The histone peptides are able to permeabilize the V. anguillarum membrane, which supports this mode of lysozyme potentiation.

Recombinant c-type and g-type lysozymes of Japanese flounder have been shown to have high bacteriolytic activity against P. piscicida and V. anguillarum but low activity against E. tarda and β-haemolytic Streptococcus sp., which are pathogenic agents of the Japanese flounder (Minagawa, Hikima, Hirono & Aoki 2001). Recombinant grouper g-type lysozyme produced in Escherichia coli expression system showed lytic activity against M. lysodeikticus, Vibrio alginolyticus, V. vulnificus, V. parahaemolyticus, V. fluviatilis, A. hydrophila and Pseudomonas fluorescens whereas mandarin g-lysozyme showed lytic activity only against M. lysodeikticus (Yin, He, Deng & Chan 2003; Sun, Wang, Xie, Gao & Nie 2006).

Nielsen and Esteve-Gassent (2006) reported that lysozyme, along with lectin, is an important non-specific immune element in the eel, preventing invasion by a wide range of pathogens. A strong lysozyme activity is detected during the early stages of eel development. Lysozyme is more active in predatory fish, which indicates their enhanced resistance and homoeostasis under changing environmental conditions in comparison with other fish species (Lukyanenko 1965a, b). The normal level of the lysozyme in the serum of fish is summarized in Table 2.

Table 2. Normal level of lysozyme in the serum of fish

Fish	Normal range/ level	Unit	Reference (s)
Seabass (Dicentrarchus labrax)	270–350	Units mL⁻¹	Obach, Quentel and Bandin Laurencin (1993)
Rainbow trout (Oncorhynchus mykiss)	600–1000	Units mL⁻¹	Verlhac et al. (1996)
Striped bass (Morone chrysops X M. saxatilis)	600–900	Units mL⁻¹	Sealey and Gatlin III (2002)
Korean rockfish (Sebastes schlegeli)	153.40–172.30	Units mL⁻¹	Jee, Masroor and Kang (2005)
Asian catfish (Clarias batrachus)	7.5–12.94	μg mL⁻¹	Kumari and Sahoo (2005a)
Grouper (Epinephelus malabaricus)	24.10	Units mL⁻¹	Lin and Shiau (2005)
Rohu (Labeo rohita)	2.85–10.80	μg mL⁻¹	Sahoo et al. (2005)
Catla (Catla catla)	2.50–8.05	μg mL⁻¹	Sahoo et al. (2005)
Mrigal (Cirrhinus mrigala)	4.65–10.25	μg mL⁻¹	Sahoo et al. (2005)
Large yellow croaker (Pseudosciaena crocea)	166.60	Units mL⁻¹	Ai, Mai, Tan, Xu, Zhang, Ma and Liufu (2006)
Pikeperch (Sander lucioperca)	46.2–48.5	mg L⁻¹	Siwicki et al. (2006)
Tilapia (Oreochromis mossambicus)	770–1000	Units mL⁻¹	Christybapita, Divyagnaneswari and Michael (2007)
Red sea bream (Pagrus major)	29.40	Units mL⁻¹	Ji, Takaoka, Jeong, Lee, Ishimaru, Seoka and Takii (2007)
Japanese eel (Anguilla japonica)	76.3	Units mL⁻¹	Ren et al. (2007)
Common carp (Cyprinus carpio)	151.28	μg mL⁻¹	Wu, Yuan, Shen, Tang, Gong, Li, Sun, Huang and Han (2007)
Olive flounder (Paralichthys olivaceus)	306	Units mL⁻¹	Yoo, Lee, Kim, Okorie, Park, Han, Choi, Kang, Sun and Bai (2007)
Yellow catfish (Horabagrus brachysoma)	7.52–11.32	μg mL⁻¹	P. K. Sahoo, unpubl. data
Bata (Labeo bata)	7.97–24.09	μg mL⁻¹	P. K. Sahoo, unpubl. data
Kalbasu (Labeo calbasu)	3.4–19.62	μg mL⁻¹	P. K. Sahoo, unpubl. data
Bronze featherback (Notopterus notopterus)	6.9–19.00	μg mL⁻¹	P. K. Sahoo, unpubl. data

Criteria for true lysozyme

According to Salton (1957) and Jolles (1969), true lysozymes have to satisfy the following criteria:

•
The enzyme lyses M. lysodeikticus cells.
•
It is readily adsorbed by chitin-coated cellulose.
•
It is a low-molecular-weight protein.
•
It is stable at acidic pH at higher temperatures and is inactivated under alkaline conditions.

Types of lysozyme, the genes involved and their expression

Lysozymes are classified into five types: chicken-type lysozyme (c-type), which includes stomach lysozyme and calcium-binding lysozyme, goose-type lysozyme (g-type), plant-type lysozyme, bacterial lysozyme and T4 phage lysozyme (phage-type) (Beintema & Terwisscha van Scheltinga 1996; Fastrez 1996; Holtje 1996; Irwin, Yu & Wen 1996; Prager & Jolles 1996; Qasba & Kumar 1997). Only the c- and g-type lysozyme have been reported in vertebrates, which have divergent amino acid compositions, molecular weights and enzymatic properties (Prager & Jolles 1996; Irwin & Gong 2003). Even though lysozyme is believed to play an important role in defence against infectious diseases in fish, only a few studies have investigated fish lysozymes. In fish, the c-type lysozyme has been reported from Japanese flounder (Paralichthys olivaceus) (Hikima, Hirono & Aoki 2000), turbot (Scophthalmus maximus) (GenBank AJ 250732), rainbow trout (Oncorhynchus mykiss) (Dautigny, Prager, Pham-Dinh, Jolles, Pakdel, Grinde & Jolles 1991), common carp (Cyprinus carpio) (Fujiki, Shin, Nakao & Yano 2000) and zebrafish (Danio rerio) (Liu & Wen 2002). Goose (g-) type lysozyme was initially identified as an antibacterial enzyme in egg whites of several avian species, and in recent years genes of its homologous have been sequenced in mammals and fish (Irwin & Gong 2003; Sun et al. 2006). In fish, the g-type lysozyme has been reported from Japanese flounder (Hikima, Minagawa, Hirono & Aoki 2001), common carp, orange-spotted grouper (Epinephelus coioides) (Yin et al. 2003) and mandarin fish (Siniperca chuatsi) (Sun et al. 2006).

The c-type lysozyme mRNA of rainbow trout (type II) is 1021 bp in length, encoding 142 amino acid residues, having a mature peptide of 129 amino acids (Dautigny et al. 1991) compared with a protein of 143 amino acids from 612 bp mRNA of Japanese flounder (Hikima, Hirono & Aoki 1997). In Japanese flounder, 15 amino acid residues comprise the signal peptide and 128 residues comprise the mature peptide. The lysozyme gene length is 3617 bp, including four exons and three introns (Hikima et al. 2000). They also found 45 novel-repeated sequences (∼1.4 kb in length) in intron 2 of the flounder c-type lysozyme gene. The rainbow trout c-type lysozyme gene is expressed in the liver and kidney (Dautigny et al. 1991) whereas the Japanese flounder c-type lysozyme gene is expressed in the head kidney, posterior kidney, spleen, brain and ovary (Hikima et al. 1997). The g-type lysozyme gene of flounder is 1252 bp, encoding 195 amino acid residues, and contains a five-exon/four-intron structure (Hikima et al. 2001). The Japanese flounder g-type gene is expressed especially in the head kidney, posterior kidney, spleen, skin, muscle, heart and brain and also in the liver, intestine, ovary and whole blood to a lesser extent. Furthermore, gene expression in the heart, intestine and whole blood increased after injection with E. tarda (Hikima et al. 2001). The chicken g-type gene is expressed only in the bone marrow and lung (Nakano & Graf 1991).

Japanese flounder c- and g-type lysozyme genes are encoded by a single copy gene (Hikima et al. 2000, 2001) as compared with tetraploid rainbow trout (Dautigny et al. 1991). When molecular evolutionary events are considered, flounder g-type lysozyme was found to be closer to the phage-type lysozyme (Hikima et al. 2001).

The g-type lysozyme cDNA of orange-spotted grouper is 585 bp in length, encoding a protein of 194 amino acids. The protein shows a 72.2% amino acid sequence identity with the flounder g-type lysozyme. The orange-spotted grouper g-type gene is expressed in the intestine, liver, spleen, anterior kidney, posterior kidney, heart, gills, muscle, stomach, brain, ovary and leucocytes (Yin et al. 2003).

The mandarin fish g-type lysozyme mRNA has 742 nucleotides (nt) in length, and contains an open reading frame of 582 nt encoding 194 amino acids, with 57 nt located in the 5′-untranslated region (UTR) and 103 in the 3′-UTR. From the first transcription initiation site, it extends 1307 nt to the end of the 3′-UTR, and contains five exons and four introns. The gene structure of mandarin fish g-type lysozyme was quite similar to its homologue in Japanese flounder, which indicates that g-lysozyme is a conserved molecule in teleosts (Sun et al. 2006). The amino acid sequence of the mandarin fish g-lysozyme shared 86% identity with orange-spotted grouper, 75% with Japanese flounder, 59% with puffer fish and 57% with zebrafish.

Factors influencing lysozyme activity in fish

Stress and infections

It is well established that the immune system of fish can be severely affected by various stress conditions. Factors such as stocking density, periodic handling, transport, water quality or the use of anaesthetics are the most common causes of stress for cultured fish in commercial intensive aquaculture (Saurabh & Sahoo 2007) and are eventually immunosuppressive. Lysozyme activity could be dependent on the degree of stress, intensity and its duration and type of stressors (Yildiz 2006). Mock and Peters (1990) reported that rainbow trout stressed by transport or acute water pollution had significantly reduced serum lysozyme levels. Caruso and Lazard (1999) reported that plasma lysozyme activity of Nile tilapia stressed by social pressure was lower than for unstressed fish. In contrast, Demers and Bayne (1997) reported that, following exposure to a handling stressor, lysozyme activity was significantly increased in rainbow trout. Enhanced serum lysozyme activity, on the other hand, was observed in carp infected with Aeromonas punctata (Siwicki & Studnicka 1987) or the protozoan Eimeria subepithelialis (Studnicka, Siwicki & Ryka 1986) and in Atlantic salmon experimentally challenged with A. salmonicida (Moyner, Roed, Sevatdal & Heum 1993). A strong increase in plasma lysozyme level was observed in both stressed and unstressed sheatfish after experimental infection with E. tarda (Caruso, Schlumberger, Dahm & Proteau 2002). The c- and g-type lysozyme mRNA levels of Japanese flounder were increased in the kidney, spleen, ovary (c-type), intestine, heart and whole blood (g-type) after fish were injected with E. tarda (Hikima et al. 2001). In carp, serum lysozyme activity increased concomitantly with the elevation of antibody titre (Vladimirov 1968). Recently, it has been reported that lysozyme possesses an antiviral function in humans (Lee-Huang, Huang, Sun, Huang, Kung, Blithe & Chen 1999).

Season, sex and stages of sexual maturity

The season, sex and sexual maturity significantly affect the lysozyme level of fish. The lysozyme level was higher in rainy and summer seasons but lower in the winter season in Indian major carp (Swain, Dash, Sahoo, Routray, Sahoo, Gupta, Meher & Sarangi 2007). A significant difference in lysozyme levels between the winter and summer season in Atlantic halibut has also been found (Bowden, Butler & Bricknell 2004). Moreover, Hutchinson and Manning (1996) reported a lower lysozyme level in dab in winter than in any other seasons of the year. This could indicate that seasonal factors might have an influence on serum lysozyme level. The low level of lysozyme in winter compared with any other seasons of the year might be one of the reasons why Indian major carps are more prone to infection in this season (Das & Das 1997).

The serum concentration of lumpsucker (Cyclopterus lumpus) lysozyme showed seasonal variations and was higher in the male than in the female (Fletcher, White & Baldo 1977). In carps, the highest level of the enzyme occurred in spawners (Studnicka et al. 1986) and in Atlantic salmon and brown trout, the lysozyme activity markedly decreased during smoltification (Muona & Soivio 1992). Schrock, Smith, Maule, Doulos and Rockowski (2001) reported seasonal variations in mucosal lysozyme levels in hatchery coho salmon and spring chinook salmon early in the parr-smoult transformation.

Water temperature

Owing to the poikilothermic nature of fish, the modulation effect of temperature on fish immune function has been well established. It has been demonstrated that the level of lysozyme in serum is affected by acclimation of fish at different temperatures. Fletcher and White (1976) reported a 70% decrease in serum lysozyme level when plaice was maintained at a low temperature (5 °C) for 3 months. A similar decrease in serum lysozyme level was observed in carp (Studnicka et al. 1986). Kumari, Sahoo, Swain, Sahoo, Sahu and Mohanty (2006) reported the lowest lysozyme level at the highest environment temperature (32.5 °C) in Asian catfish Clarias batrachus during summer. Similarly, Japanese eels maintained at 15 °C showed greater serum lysozyme activity compared with those maintained at 20–30 °C (Kusuda & Kitadai 1992). Similarly, one study revealed that the lysozyme level in Sparus aurata and Oreochromis niloticus was less sensitive to seasonal or temperature changes (Hernandez & Tort 2003; Dominguez, Takemura & Tsuchiya 2005), whereas a clear reduction in the lysozyme level was marked in S. aurata at a lower temperature (Tort, Rotllant, Liarte, Acerete, Hernandez, Ceulemans, Coutteau & Padros 2004). In general, a positive correlation between plasma lysozyme activity and water temperature has been reported in many fish species (Langston, Hoare, Stefansson, Fitzgerald, Wergeland & Mulcahy 2002; Watts, Munday & Burke 2002).

Environmental salinity

Several studies demonstrated that changes in environmental salinity modulate the lysozyme level of fish (Dominguez et al. 2005; Taylor, Needham, North, Morgan, Thompson & Migaud 2007). An increase in circulating lysozyme activity at higher salinity was observed in brown trout (Marc, Quentel, Severe, Le Bail & Boeuf 1995), rainbow trout (Yada, Azuma & Takagi 2001) and Nile tilapia (Dominguez et al. 2005). On the contrary, the plasma lysozyme activity was not changed in Mozambique tilapia following the transfer of fish from freshwater to seawater (Yada, Uchida, Kajimura, Azuma, Hirano & Grau 2002). Moreover, it is reported that changes in environmental salinity are accompanied by alterations in endogenous hormones for euryhaline species (Sakamoto, McCormick & Hirano 1993). One study revealed an increase in the plasma growth hormone (GH) level and lysozyme activity in seawater-transferred brown trout and suggested that GH stimulates macrophage function and increases non-specific immune potential. It is therefore likely that environmental salinity stimulates increased GH secretions to modulate the lysozyme level of euryhaline fish.

Low pH and sedimentation

pH is a measure of hydrogen ion concentration in water and indicates how much water is acidic or basic. Water pH affects the lysozyme activity of fish. Dominguez et al. (2005) reported enhanced lysozyme activity in Nile tilapia when the fish were kept in acidic water for 2 weeks. Similarly, the presence of suspended solids in the culturable water also affected the lysozyme level of fish. One study revealed that the lysozyme level in O. niloticus was enhanced at the suspended sediments' level of 2000 mg L⁻¹ but no change in the lysozyme activity was recorded in the suspended sediments' level of 20 and 200 mg L⁻¹ (Dominguez et al. 2005). Therefore, it is likely that higher concentrations of suspended solids in the water alter the lysozyme levels in fish.

Nutrition

The interaction between nutrition and defence mechanisms in fish has long been known, but this relationship is far more complex than considered originally (Siwicki, Zakes, Fuller, Nissen, Trapkowska, Glabski, Kowalska, Kazun & Majewska 2006). Nutritional support plays an important role in maintaining optimum the health conditions of organisms by providing the building blocks of non-specific cellular and humoral immunity and thus protection against a wide variety of diseases (Kumar, Saurabh, Sahu & Pal 2005; Saurabh & Mohanta 2006). Certain nutrients can be supplemented in the feed to modulate the lysozyme activity of fish. Enhanced lysozyme activity has been reported by oral administration of glucan in many fish including Atlantic salmon (Engstad & Robertsen 1993), rohu (Sahoo & Mukherjee 2001), snapper (Cook, Hayball, Hutchinson, Nowak & Hayball 2003), African catfish (Yoshida, Kruger & Inglis 1995) and Asian catfish (Kumari & Sahoo 2006). Kumari and Sahoo (2006) reported that β-1,3 glucan at a rate of 0.1% in the feed of C. batrachus activates the lysozyme activity and provides protection against motile aeromonad septicaemia caused by A. hydrophila. Similarly, stimulating effects of vitamin C and E on lysozyme activity have been demonstrated in various fish species (Waagbo, Glette, Raa Nilsen & Sandnes 1993; Roberts, Davies & Pulsford 1995; Verlhac, Gabaudan, Obach, Schuep & Hole 1996; Sahoo & Mukherjee 2002a, b, 2003).

Aquatic toxicants

Aquatic toxicants are a diverse group of substances that have two general properties: they are discharged into the environment, and they have the potential to impact on ecosystems at relatively low concentrations (Bols, Brubacher, Ganassin & Lee 2001). Fish are the most sensitive aquatic organisms and show a wide range of responses even towards minor changes in their environment. Exposure to heavy metals modulates lysozyme levels but the nature of the modulation can be complex. Serum lysozymes activity was elevated in rainbow trout exposed for 30 days to cadmium, mercury or zinc and to mixtures of these metals (Sanchez-Dardon, Voccia, Hontela, Chilmonczyk, Dunier, Boermans, Blakely & Fournier 1999). Fletcher (1986) reported that sub-lethal concentrations of inorganic mercury reduce the circulatory levels of lysozyme in the plaice. When rainbow trout were injected with diesel-oil drilling mud, the serum lysozyme level was reduced at a dose of 0.6 mL kg⁻¹ but not at higher doses (Tahir & Secombes 1995). Serum lysozyme levels were reduced in dab caught near an oil-tanker accident after experimental exposure of dab to oil-contaminated sediments (Tahir, Fletcher, Houlihan & Secombes 1993). In rainbow trout maintained for 28 days in creosote microcosms, plasma lysozyme levels were reduced and the reduction increased with increasing doses of creosote (Karrow, Boermans, Dixon, Hontella, Solomon, Whyte & Bols 1999). On the contrary, insecticide trichlorophon slightly reduced lysozyme activity in the carp (Siwicki, Cossarini-Dunier, Studnicka & Demael 1990). Similarly, reduced serum lysozyme in Korean rockfish was reported following exposure to synthetic pyrethroids (Jee et al. 2005). Nakayama, Kurokawa, Harino, Kawahara, Miyadai, Seikai and Kawai (2007) reported that tributylin, which is used for antifouling paint of ship's hulls and fishing nets, inhibits lysozyme activity in Japanese flounder. These studies suggest that the level of lysozyme in fish serum is a convenient parameter for monitoring the potential impact of environmental hazards on fish innate immunity.

Immunostimulants and lysozyme

An immunostimulant is a chemical, drug, naturally occurring compound, stressor or action that acts on the non-specific defence mechanisms or the specific immune response of the host to handle pathogens (Sahoo 2004, 2007). Immunostimulants has to be given alone to activate non-specific defence mechnanisms as well as heighten a specific immune response. A large number of immunostimulants have been reported to increase serum lysozyme levels in fish that may be due to either an increase in the number of phagocytes secreting lysozyme or due to an increase in the amount of lysozyme synthesized per cell (Engstad, Robertsen & Frivold 1992; Kumari & Sahoo 2006). Changes in lysozyme activity are considerably influenced by the potency and the type of immunostimulants to which fish are exposed. An increase in lysozyme following immunostimalation has been demonstrated in various fish species, and its association with increasing protection against a variety of fish diseases is summarized in Table 3. Another advantage of immunostimulants is their use as adjuvants for fish vaccination. Thus, the use of immunostimulants is an effective means of increasing the immunocompetency and disease resistance of fish.

Table 3. Effects of immunostimulants on the lysozyme activity of fish and its protection against pathogens

Immunostimulants	Lysozyme activity	Fish	Protection against pathogens	Reference (s)
Vitamin C	Increase	Salmon	A. salmonicida	Waagbo, Sandnes, Lie and Nilsen (1993)
Levamisole	Increase	Turbot	V. anguillarum	Skjermo, Defoort, Dehasque, Espevik, Olsen, Skjak-Brfek, Sorgeloos and Vadstein (1995)
Soybean products	Increase	Atlantic salmon	A. salmonicida	Krogdahl and Roed (2000)
Vitamin E	Increase	IMC	A. hydrophila & E. tarda	Sahoo and Mukherjee (2002a, b)
IL-1β	Increase	Trout	A. salmonicida	Hong, Peddie, Campos-Perez, Jou and Secombes (2003)
Traditional Chinese medicine	Increase	Yellow croaker	V. alginolyticus	Jian and Wu (2003)
Lactoferrin	Increase	Catfish	A. hydrophila	Kumari, Swain and Sahoo (2003)
Triiodothyronine	Increase	IMC	A. hydrophila	Sahoo (2003)
Vitamin C	Increase	IMC	A. hydrophila & E. tarda	Sahoo and Mukherjee (2003)
Vitamin C	Increase	Large yellow croaker	V. harveyi	Ai et al. (2006)
Chitosan	Increase	Carp	A. hydrophila	Gopalakannan and Arul (2006)
Levamisole	Increase	Carp	A. hydrophila	Gopalakannan and Arul (2006)
Glucan	Increase	Catfish	A. hydrophila	Kumari and Sahoo (2006)
Tuftsin	Increase	IMC	A. hydrophila & E. tarda	Misra, Das, Mukherjee and Meher (2006)
Herbal materials Achyranthes aspera seed	Increase	IMC	A. hydrophila	Vasudeva Rao, Das, Jyotyrmayee and Chakrabarti (2006)
Carbohydrate with n-3 PUFA	Increase	IMC	A. hydrophila	Misra, Sahu, Pal, Xavier, Kumar and Mukherjee (2006)
β-hydroxy-β-methyl butyrate (HMB)	Increase	Pikeperch	A. salmonicida	Siwicki et al. (2006)
A3α-Peptodoglycan	Increase	Japanese flounder	V. anguillarum	Zhou, Song, Huang and Wang (2006)
Glucan	Increase	Large yellow croaker	V. harveyi	Ai, Mai, Zhang, Tan, Zhang, Xu and Li (2007)
CpG oligodeoxynucleotide (ODN)	Increase	Rainbow trout	A. salmonicida	Carrington and Secombes (2007)
Sodium alginate and iota-carrageenan	Increase	Orange-spotted Grouper	V. alginolyticus	Cheng, Tu, Chen, Nan and Chen (2007)
Eclipta alba (medicinal plants)	Increase	Tilapia	A. hydrophila	Christybapita et al. (2007)
Solanum trilobatum leaf (medicinal plant)	Increase	Tilapia	A. hydrophila	Divyagnaneswari, Christybapita and Michael (2007)
AquaImmu (herbal material)	Increase	IMC	A. hydrophila	Kumari, Sahoo and Giri (2007)
Levan	Increase	Common carp	A. hydrophila	Rairakhwada, Pal, Bhathena, Sahu, Jha and Mukherjee (2007)
Allium sativum	Increase	IMC	A. hydrophila	Sahu, Das, Mishra, Pradhan and Sarangi (2007)
Magnifera indica kernel	Increase	IMC	A. hydrophila	Sahu, Das, Pradhan, Mohapatra, Mishra and Sarangi (2007)
Bovine lactoferrin	Increase	Nile tilapia	Streptococcus iniae	Welker et al. (2007)
β-1,3-glucan and feeding stimulants	Increase	Olive flounder	E. tarda	Yoo et al. (2007)

A. salmonicida, Aeromonas salmonicida; E. tarda, Edwardsiella tarda; A. hydrophila, Aeromonas hydrophila; V. alginolyticus, Vibrio alginolyticus.

Probiotics and lysozyme

The term probiotic is derived from the Greek words Pro (favour) and Bios (life). Probiotics are defined as microbial cell preparations or components of microbial cells that have a beneficial effect on the health and well-being of the host (Salminen, Ouwehand, Benno & Lee 1999; Saurabh, Choudhary & Sushma 2005). To date, little is known about probiotic effects on lysozyme activity. However, Panigrahi, Kiron, Kobayashi, Puangkaew, Satoh and Sugita (2004) showed significantly higher lysozyme activity in rainbow trout fed with Lactobacillus rhamnosus. Similarly, Kim and Austin (2006) recorded high gut mucosal lysozyme activity in fish fed with Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33. Taoka, Maeda, Jo, Kim, Park, Yoshikawa and Sakata (2006) also reported an enhanced lysozyme level in tilapia fed with live and dead probiotic cell and recorded a high survival rate when challenged with E. tarda.

Lysozyme and disease resistance

Selection for disease resistance based on variation in the lysozyme activity of serum seems to be considerably complicated as it is highly influenced by the immune status of fish (Roed, Fevolden & Fjalestad 2002). A significantly higher mortality in high lysozyme lines of rainbow trout challenged with A. salmonicida compared with the low lysozyme lines was found (Roed et al. 2002). Similarly, Lund, Gjedrem, Bentsen, Eide, Larsen and Roed (1995) observed an apparently negative genetic association between lysozyme activity and survival rate in full-sib groups of Atlantic salmon when challenged with A. salmonicida. Fevolden, Roed and Gjerde (1994) reported negative genetic correlations between serum lysozyme activity in stressed Atlantic salmon and survival in challenge tests with furunculosis and bacterial kidney disease. Roed, Larsen, Linder and Refstie (1993) also observed a negative phenotypic correlation between serum lysozyme activity of Atlantic salmon and survival in a challenge test with vibriosis. On the other hand, Sahoo, Meher, Mahapatra, Saha, Jana and Reddy (2004) did not find any significant correlation between the serum lysozyme activity and survival rate in different fullsib families of Indian major carp, Labeo rohita when challenged with A. hydrophila. Further, high heritability (0.6–0.7) of lysozyme activity at an incubation temperature of 30 °C and intermediate heritability (∼0.3) at 15 °C were observed in Nile tilapia (O. niloticus) by Chiayvareesajja, Roed, Eknath, Danting, De Vera and Bentsen (1999). The high heritability of serum lysozyme activity and the significant negative genetic association between this trait and survival rate suggest that serum lysozyme activity may be a promising candidate trait for indirect selection to improve the survival rate of fish. According to the negative association between the trait and survival rate, broodstock should be selected among individuals and families with low serum lysozyme activity. Furthermore, a linkage between resistance to furunculosis and low lysozyme activity in Atlantic salmon (Lund et al. 1995); resistance to Vibrio sp. and plasma lysozyme activity in coho salmon, chinook salmon and tilapia (Balfry, Heath & Iwama 1997; Balfry & Iwama 2004); and serum lysozyme activity and resistance to A. salmonicida, R. salmoninarum and V. salmonicida infections in salmonids (Lund et al. 1995) have been observed.

Conclusion

Lysozyme is a widely distributed hydrolase, which likely plays an important role in the bio-defence system. This enzyme has antiviral, antibacterial and anti-inflammatory properties. Lysozyme is widely distributed in fish at their body surface, skin, gill, intestinal tract and serum as a protection factor against bacterial infection. Fish lysozymes possess a high potential for bacteriocidal or bacteriolytic activity against Gram-positive and Gram-negative bacteria. Season, sex, sexual maturity, water temperature, nutritional substances, stress and infection affect serum the lysozyme level of fish. Immunization of fish with antigen leads to an increase in both antibody and lysozyme titres, with the intensity of antibody formation being directly proportional to the activity of the lysozyme. Such an increase in the enzyme level can reflect changes in the white cell population during the development of the immune response. Thus, infection, which brings about changes in the numbers of leucocytes, may affect lysozyme concentration, and estimation of lysozyme may be of diagnostic value to determine the disease status of fish. Lysozyme has also been established as an immune marker in selective breeding of fish for bacterial disease resistance. These findings suggest that further elucidation of lysozyme activity against invading microorganisms is important for a better understanding of host–pathogen interaction and immunity.

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Lysozyme: an important defence molecule of fish innate immune system

Abstract

Introduction

Lysozyme and its function

Criteria for true lysozyme

Types of lysozyme, the genes involved and their expression

Factors influencing lysozyme activity in fish

Stress and infections

Season, sex and stages of sexual maturity

Water temperature

Environmental salinity

Low pH and sedimentation

Nutrition

Aquatic toxicants

Immunostimulants and lysozyme

Probiotics and lysozyme

Lysozyme and disease resistance

Conclusion

References

Citing Literature

References

Information

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Lysozyme: an important defence molecule of fish innate immune system

Abstract

Introduction

Lysozyme and its function

Criteria for true lysozyme

Types of lysozyme, the genes involved and their expression

Factors influencing lysozyme activity in fish

Stress and infections

Season, sex and stages of sexual maturity

Water temperature

Environmental salinity

Low pH and sedimentation

Nutrition

Aquatic toxicants

Immunostimulants and lysozyme

Probiotics and lysozyme

Lysozyme and disease resistance

Conclusion

References

Citing Literature

References

Related

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