sakai(1999)

Ž .Aquaculture 172 1999 63–92

Current research status of fish immunostimulants

Masahiro Sakai )

Faculty of Agriculture, Miyazaki UniÕersity, Miyazaki 889-21, Japan

Accepted 30 October 1998

Abstract

Immunostimulants are valuable for the control of fish diseases and may be useful in fishculture. The immunostimulatory effects of glucan, chitin, lactoferrin and levamisole for fish andshrimp have been reported. Nutritional factors such as Vitamins B and C, growth hormone andprolactin have also been reported to be immunostimulators. These immunostimulants mainlyfacilitate the function of phagocytic cells and increase their bactericidal activities. Severalimmunostimulants also stimulate the natural killer cells, complement, lysozyme and antibodyresponses of fish. The activation of these immunological functions is associated with increasedprotection against infectious disease. Resistance to bacterial pathogens such as Vibrio anguil-larum, V. salmonicida, Aeromonas salmonicida, Yersinia rukeri and Streptococcus spp. and toparasitic infections such as white spot disease can be increased by administration of immunostimu-lants, but not to intracellular pathogens such as Renibacterium salmoninarum and Pasteurellapiscicida. The most effective method of administration of immunostimulants to fish is byinjection. Oral and immersion methods have also been reported, but the efficacy of these methodsdecreases with long-term administration. Overdoses of several immunostimulants induce immuno-suppression in fish. The side effects of immunostimulants have not been well-studied. Growth-promoting activity has been noted in fish or shrimp treated with glucan or lactoferrin. Immunos-timulants can overcome immune suppression by sex hormones. Thus, the influence of immunos-timulants in mature fish should be studied. In conclusion, immunostimulants can reduce the lossescaused by disease in aquaculture; however, they may not be effective against all diseases. For theeffective use of immunostimulants, the timing, dosages, method of administration and thephysiological condition of fish need to be taken into consideration. q 1999 Elsevier Science B.V.All rights reserved.

Keywords: Immunostimulant; Non-specific immunity; Macrophage; Fish; Aquaculture

) Fax: q81-985-58-2884

0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 98 00436-0

( )M. SakairAquaculture 172 1999 63–9264

1. Introduction

Fish culture is an important industry. Various kinds of marine and freshwater fishhave been cultured and the worldwide production of cultured fish increases every year.Fishes are usually cultured in enclosed spaces such as ponds or net cages and effortshave been made to increase productivity per unit space. Overcrowding tends toadversely affect the health of cultured fish. These conditions tend to produce poorphysiological environment for fish and increase susceptibility to infections.

Various chemotherapeutics have been used to treat bacterial infections in culturedfish for about the last 20 years. However, the incidence of drug-resistant bacteria has

Ž .become a major problem in fish culture Aoki, 1992 . Vaccination is a useful prophy-laxis for infectious diseases of fish and is already commercially available for bacterialinfections such as vibriosis, redmouth disease and furunculosis and for viral infectionsuch as IPN. Vaccination may be the most effective method of controlling fish disease.Furthermore, the development of vaccines against intracellular pathogens such asRenibacterium salmoninarum has not so far been successful. Therefore, the immediatecontrol of all fish diseases using only vaccines is impossible.

Immunostimulants increase resistance to infectious disease, not by enhancing specificimmune responses, but by enhancing non-specific defense mechanisms. Therefore, thereis no memory component and the response is likely to be of short duration. Use of theseimmunostimulants is an effective means of increasing the immunocompetency anddisease resistance of fish. Research into fish immunostimulants is developing and manyagents are currently in use in the aquaculture industry.

Use of immunostimulants, in addition to chemotheraputic agents and vaccines, hasbeen widely accepted by fish farmers. Many questions about the efficacy of immunos-

Žtimulants from users still remain for example, whether immunostimulants can protect.against all infectious diseases . In this review, the current status of immunostimulant

research will be discussed.

2. Fish defense system enhancement by immunostimulants

Ž .The fish defense system Iwama and Nakanishi, 1996 is basically similar to thatdescribed in mammals. For cellular defense systems, teleosts have phagocytic cells

Ž .similar to macrophages, neutrophils, and natural killer NK cells, as well as T and Blymphocytes. Teleosts also have various humoral defense components such as comple-

Ž .ment classical and alternative pathways , lysozyme, natural hemolysin, transferrin andŽC-reactive protein. The existence of cytokines interferon, interleukin 2, macrophage

. Ž .activating factors has also been reported Secombes et al., 1996 .Fishes treated with immunostimulants usually show enhanced phagocytic cell activi-

w Ž .ties glucan Yano et al., 1989; Chen and Ainsworth, 1992; Jørgensen et al., 1993a ,Ž . Ž . Žlactoferrin Sakai et al., 1993 , levamisole Kajita et al., 1990 , FK-565 Kitao and

. Ž . Ž .xYoshida, 1986 , chitin Sakai et al., 1992 , EF203 Yoshida et al., 1993 . The activitiesof phagocytic cells can be detected by phagocytosis, killing and chemotaxis. Enhance-ment of pathogen killing is most important in the macrophages of fish treated with

( )M. SakairAquaculture 172 1999 63–92 65

immunostimulants. Killing mechanisms of macrophages can be broadly categorized asoxygen-dependent or oxygen-independent. Oxygen-dependent killing mechanisms as

Ž .mediated by reactive oxygen species ROS can be detected by the chemiluminescenceŽ . Ž .Kajita et al., 1990 and the NBT test Sakai et al., 1995e . Recently, the importance of

Ž .reactive nitrogen species RNS in macrophage killing, especially in the killing ofŽ .intracellular pathogens, has been indicated Nathan and Hibbs, 1991 . However, there is

no report of the activation of RNS in the macrophages of fish treated with immunostim-ulants.

Lymphocytes are also activated by immunostimulants. Immunostimulants enhancethe mitogen activities induced by concanavalin A or lipopolysaccharides and produce

Ž .macrophage activating factors Hardie et al., 1991; Siwicki et al., 1996 . ComplementŽ .activity can also be activated by several immunostimulants. Li and Lovell 1985 and

Ž .Hardie et al. 1991 reported that fish given the large amounts of vitamin C increasedlevels of complement activity. Atlantic salmon injected with yeast glucan also showed

Ž .increased complement activities Engstad et al., 1992 . Lysozyme activity is alsoŽinfluenced by the administration of immunostimulants Engstad et al., 1992; Jørgensen

. Ž . Ž .et al., 1993a . NK cells can be activated by growth hormone GH Kajita et al., 1992bŽ .and levamisole Kajita et al., 1990 .

Antibody production can also be enhanced by many immunostimulants. Channelcatfish injected with yeast glucan responded to subsequent Edwardsiella ictaluri immu-

Žnization with higher serum antibody titers relative to controls Chen and Ainsworth,. Ž .1992 . Aakre et al. 1994 reported that Atlantic salmon injected with a mixture of

Aeromonas salmonicida bacterin and yeast glucan showed enhanced antibody responses.The enhancement of antibody responses to A. salmonicida bacterin was also reported

Ž . Žwith administration of vitamin C Thompson et al., 1993 and FK-565 Kitao et al.,.1987 .

3. Immunostimulants studied in fish and shellfish

Table 1 lists those immunostimulants reported to be effective in fish and shellfish. Inthis review, immunostimulants include chemical agents, bacterial components, poly-saccharides, animal or plant extracts, nutritional factors and cytokines.

3.1. Synthetic chemicals

3.1.1. LeÕamisoleLevamisole is an anthelminthic used for the treatment of nematode infections in man

and animals. Incidental observations suggested a state of enhanced resistance to variouskinds of infection upon treatment. Levamisole in mammals enhances the metabolic andphagocytic activities of neutrophils, and increases the number of phagocytes and

Ž .leucocytes and the level of lysozyme in serum Symoens and Rosenthall, 1977 . AnimalŽ .studies also suggested a stimulatory effect on tumor immunity Fidler and Spitler, 1975 .

Coho salmon, Oncorhynchus kisutch, injected with levamisole mixed with modifiedŽ .Freund’s complete adjuvant MFCA showed increased resistance to A. salmonicida


Table 1Immunostimulants used in fish and shrimp

Synthetic chemicalsLevamisoleFK-565

Ž .MDP Muramyl dipeptide

Biological substancesŽ .1 Bacterial derivativesb-glucan

Ž .Peptidoglucan BreÕibacterium lactofermentum; Vibrio sp.FCAEF203

Ž .LPS lipopolysaccharideClostridium butyricum cellsAchromobacter stenohalis cells

Ž .Vibrio anguillarum cells Vibrio vaccineŽ .2 PolysaccharidesChitinChitosanLentinanSchizophyllanOligosaccharideŽ .3 Animal and plant extracts

Ž .Ete TunicateŽ .Hde Abalone

Firefly squidŽ .Quillaja saponin soap tree

Ž .Glycyrrhizin licoriceŽ .4 Nutritional factorsVitamin CVitamin EŽ .5 Hormones, cytokines and othersLactoferrinInterferonGrowth hormoneProlactin

Ž . Ž .Olivier et al., 1985 and Siwicki 1987, 1989 reported that carp injected withlevamisole showed enhanced phagocytic activity and myeloperoxidase activity in neu-

Ž .trophils, increased leucocyte numbers and serum lysozyme levels. Kajita et al. 1990reported that rainbow trout injected with levamisole showed increased protection againstVibrio anguillarum, caused by the enhancement of non-specific immune responses suchas phagocytic activity, chemiluminescance responses of leucocytes and NK cell activi-ties. Concentration of complement also increased in levamisole-injected rainbow trout.

Ž .Levamisole can stimulate immune responses in vitro. Siwicki et al. 1990 reportedthat in vitro immunization of spleen cells cultured with levamisole enhanced phagocytic

Žactivity, the NBT reaction and increased the number of plaque forming cells antibody-.producing cells against Yersinia ruckeri O-antigen. The immunostimulatory effects of


levamisole by oral and immersion administration have also been reported. SiwickiŽ .1989 reported that oral administration of levamisole increased the number of leuco-cytes, lysozyme activities in serum and the stimulated NBT reduction and phagocytic

Ž .index of phagocytic cells. Baba et al. 1993 reported that carp immersed in a levamisoleŽ .bath 10 mgrml, 24 h showed enhanced resistance against Aeromonas hydrophila, and

its phagocytes increased chemotactic ability, phagocytic activity and chemilumines-Ž .cence. Jeney and Anderson 1993a also showed that bathing rainbow trout for 30 min in

Ž .levamisole solution 5 mgrml before a 2 min bath in A. salmonicida O-antigenŽbacterin elevated both the non-specific defense mechanisms phagocytic activity and

. Ž .index and specific antibody titers . Although the immunostimulating efficacy oflevamisole has been reported in fish, the optimum doses should be considered. Kajita et

Ž . Ž .al. 1990 indicated that rainbow trout injected with high doses 5 mgrkg of levamisoledid not show stimulated chemiluminescence responses of head kidney leucocytes,

Ž .compared with fish injected with the optimum dose 0.1–0.5 mgrkg . The same effectŽ .was also observed in vitro. Siwicki et al. 1990 demonstrated that in vitro immunization

with Y. ruckeri O-antigen cultured with 50 mgrml of levamisole did not stimulate theantibody forming cells in the spleen of rainbow trout, and the antibody production wascompletely suppressed by the high doses of levamisole. The immunostimulating effectsof levamisole have been clearly shown in fish, and this agent can be administered byvarious methods. However, the safety of levamisole still remains to be determined,especially when administered high doses.

3.1.2. FK-565Ž Ž . Ž . .FK-565 heptanoyl-y-D-glutamyl- L -meso-diaminopimelyl- D -alanine is a peptide

Ž .related to lactoyl tetrapeptide FK-156 isolated from cultures of Streptomyces oli-ŽÕaceogriseus and has been shown to be active against microbial infection in mice Mine

. Ž .et al., 1983 . Kitao and Yoshida 1986 applied FK-565 to fish and demonstrated that theinjection of FK-565 in to rainbow trout increased their resistance to A. salmonicida,following the activation of phagocytic cells. The activation of phagocytic cells was also

Ž .observed in yellowtail Yoshida, personal communication . Numbers of splenic anti-body-producing cells and humoral antibody titers can be elevated during immunizationin rainbow trout when Y. ruckeri or A. salmonicida O-antigen preparations are mixed

Ž .with FK-565 Kitao et al., 1987 .

3.2. Bacterial deriÕatiÕes

3.2.1. MDPŽ .Ž .Muramyl dipeptide MDP N-acetyl-muramyl-L-alanyl-D-isoglutamine , derived from

Mycobacterium, elicits an immunostimulatory effect; e.g., the activation of macrophages,Ž .B lymphocytes and alternative pathway of complement in mice. Olivier et al. 1985

reported that coho salmon injected with a mixture of MDP and modified Freund’sincomplete adjuvant should a 47-fold increase in resistance to A. salmonicida. Kodama

Ž .et al. 1993 reported that intraperitoneal injection of rainbow trout with MDP–Lysincreased the phagocytic activities, respiratory burst and migration activities of kidneyleucocytes as well as resistance of the fish to A. salmonicida challenge. Furthermore,


they demonstrated that sera of fish injected with MDP–Lys could stimulate colonyŽ .formation of kidney cells Kodama et al., 1994 .

3.2.2. LPSŽ .LPS lipopolysaccharide is a cell wall component of gram-negative bacteria. LPS

can stimulate B cell proliferation, and, LPS injected into red sea bream Pagrus majorŽ .has been demonstrated to enhance macrophage phagocytic activity Salati et al., 1987 .

Ž .MacArthur et al. 1985 reported that plaice, Pleuronectes platessa, injected with LPSshowed increased macrophage migratory activity. In vitro, LPS stimulates phagocytosis

Žand the production of superoxide anions in Atlantic salmon macrophages Solem et al.,.1995 . Similarly, LPS stimulates the production of macrophage activating factor in

Ž .goldfish lymphocytes Neumann et al., 1995 and the production of interleukin 1 likeŽ .molecules in catfish monocytes Clem et al., 1985 .

3.2.3. FCAŽ .Freund’s complete adjuvant FCA , a mineral oil adjuvant containing killed My-

cobacterium butyricum, enhances immune responses and increases the efficacy ofŽ . Ž .vaccination in fish e.g., Paterson and Fryer, 1974 . Olivier et al. 1985 reported that

Ž .coho salmon, O. kisutch, injected with FCA increased protection 450 times in LD50

against A. salmonicida challenge. This increased protection was also seen against A.hydrophila and V. ordalii. Immunologically, they demonstrated that the activation of

Ž .macrophages phagocytosis and killing in fish injected with FCA caused increasedŽ . Ž .resistance to A. salmonicida Olivier et al., 1986 . Adams et al. 1988 also reported that

FCA-injected rainbow trout appeared to show increased protection against furunculosis,Ž .vibriosis and redmouth disease. Kajita et al. 1992a confirmed that rainbow trout

injected with FCA showed increases in respiratory burst, phagocytic and NK cellactivity of leucocytes and protection against V. anguillarum infection. However,

Ž .Kawakami et al. 1998 showed that yellowtail injected with FCA did not showincreased resistance to P. piscicida infection, although the adjuvant effect of FCA on aP. piscicida vaccine was observed in fish.

3.2.4. Vibrio bacterinV. anguillarum bacterin is the most successful vaccine for salmonid fish, and this

efficacy is seen following administration by injection, oral dosing and immersionŽ .methods. Sakai et al. 1995c reported that rainbow trout immersed in V. anguillarum

Ž .bacterin solution showed increased protection 12 times in LD to Streptococcus sp.50Ž .infection. Norqvist et al. 1989 also showed that vaccination of rainbow trout with

attenuated V. anguillarum induced protection against A. salmonicida challenge. How-ever, no immunostimulation of V. anguillarum bacterin was seen in yellowtail infected

Ž .with P. piscicida Kawakami et al., 1998 . The immunostimulant effects of vibrioŽ .bacterins were also reported in Kuruma prawns Penaeus japonicus. Itami et al. 1989

reported that prawns injected or immersed with the formalin-killed vibrio bacterinexperienced reduced mortalities when they were challenged by Vibrio injection 30 days

Ž .later. Horne et al. 1995 also reported the efficacy of vibrio vaccines in black tigershrimp. The migration of hemocytes treated with vibrio bacterin increased, compared to


Ž .non-treated controls Itami et al., 1989 . This fact suggested the immunostimulation ofvibrio bacterin in prawn and shrimp, as invertebrates do not have specific immuneresponses. It is still unknown what components of V. anguillarum cells stimulatenon-specific immune responses. However, as bacterial LPS stimulated non-specificimmunity, especially macrophage activation, V. anguillarum LPS may act as animmunostimulant.

3.2.5. A. stenohalis and C. butyricumA. stenohalis is a gram-negative aerobic organism which has been isolated from sea

Žwater. The LPS of this bacteria activates mouse macrophages and B-lymphocytes Isogai. Ž . Ž .et al., 1989 and shows anti-tumor effects Chang, 1966 . Kawahara et al. 1994

reported that white-spotted char, SalÕelinus leucomaenis, injected with inactivated A.stenohalis showed enhanced chemiluminescent responses of kidney cells, complementactivation and increased protection against A. salmonicida challenge.

A butyric acid bacterium, C. butyricum, has been used clinically to prevent distur-bances of microbial flora in the human intestine, and oral administration of the spores ofthis bacterium ameliorate diarrhoea, constipation and abdominal distension in man. C.butyricum shows immunostimulatory effects such as stimulation of macrophages and

Ž .NK cells and enhances protection against Candida infection Young et al., 1987 . SakaiŽ .et al. 1995a,b showed enhancement of resistance to vibriosis in rainbow trout by oral

administration of C. butyrium bacterin mediated by leucocyte activation, includingphagocytosis and increased superoxide anion production.

3.2.6. Chitin and chitosanChitin is a polysaccharide forming the principal component of crustacean and insect

Ž .exoskeletons and the cell walls of certain fungi. Sakai et al. 1992 reported that rainbowtrout injected with chitin showed stimulated macrophage activities and an increasedresistance to V. anguillarum infection. Yellowtail injected with chitin alone also showedincreased protection against P. piscicida challenge which continued until 45 days after

Ž .treatment Kawakami et al., 1998 . Compared with the increase in non-specific protec-tive immunity in yellowtail injected with chitin, chitin did not show adjuvant effectsŽ .Kawakami et al., 1998 , as had been was demonstrated in mice and guinea pigsŽ .Nishimura et al., 1985 .

Chitosan, de-N-acetylated chitin, also showed immunostimulatory effects. Brooktrout, SalÕelinus fontinalis, injected or immersed in chitosan solution showed increased

Ž .protection against A. salmonicida infection Anderson and Siwicki, 1994 as didŽ .rainbow trout administered chitosan orally Siwicki et al., 1994 . Rainbow trout treated

with chitosan by injection or immersion showed increases in immunological parametersin the blood such as NBT, potential killing activity, myeloperoxidase and total Ig

Ž .concentration Anderson et al., 1995 .

3.2.7. EF203EF203 is the fermented product of chicken eggs. The oral administration of EF203 to

rainbow trout stimulates the activity of leucocytes such as phagocytosis and chemilumi-Žnescence, and increases protection against Streptococcus infection Yoshida et al.,

()

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Table 2Immunostimulant effects in fish and shrimp

Agent Authors Fish Administration Results Resistance to pathogens

Ž .Levamisole Siwicki 1987 carp ip phagocytosis ≠

Ž .Siwicki 1989 carp oral NBT ≠Ž .Siwicki et al. 1990 trout in vitro phagocytosis ≠

NBT ≠Ž .Kajita et al. 1990 trout ip phagocytosis ≠ V. anguillarum ≠

CL ≠

complement ≠Ž .Jeney and Anderson 1993a trout im A. salmonicida ≠

Ž .Baba et al. 1993 carp im phagocytosis ≠ A. hydrophila ≠

CL ≠Ž .FK-565 Kitao and Yoshida 1986 trout ip phagocytosis ≠ A. salmonicida ≠

Ž .Kitao et al. 1987 trout in vitro antibody ≠Ž .MDP Kodama et al. 1993 trout ip phagocytosis ≠ V. anguillarum ≠

CL ≠Ž .LPS Clem et al. 1985 catfish in vitro IL1 ≠

Ž .MacArthur et al. 1985 plaice ip macrophage migration ≠Ž .Salati et al. 1987 red sea bream ip phagocytosis ≠

Ž .Neumann et al. 1995 goldfish in vitro MAF ≠Ž .Solem et al. 1995 salmon in vitro phagocytosis ≠

NBT ≠Ž .Dalmo and Seljelid 1995 salmon in vitro phagocytosis ≠

NBT ≠Ž .FCA Olivier et al. 1985 coho ip A. salmonicida ≠

A. hydrophila ≠

V. ordarii ≠Ž .Olivier et al. 1986 brook ip phagocytosis ≠Ž .Adams et al. 1988 trout ip A. salmonicida ≠

V. anguillarum ≠

Y. ruckeri ≠Ž .Kajita et al. 1990 trout ip V. anguillarum ≠

Ž .Kawakami et al. 1998 yellowtail ip P. piscicida ™

()

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Ž .Vibrio Norqvist et al. 1989 trout im A. salmonicida ≠Ž .bacteria Sakai et al. 1995c trout im Enterococcus seriolicida ≠Ž .Itami et al. 1989 prawn ip Vibrio sp. ≠

imŽ .Horne et al. 1995 shrimp ip Vibrio sp. ≠

imoral

Ž .C. butyricum Sakai et al. 1995a,b trout oral phagocytosis ≠ V. anguillarum ≠

NBT ≠Ž .A. stenohalis Kawahara et al. 1994 char ip CL ≠ A. salmonicida ≠

complement ≠Ž .Chitin Sakai et al. 1992 trout ip phagocytosis ≠ V. anguillarum ≠

lysozyme ™Ž .Chitosan Siwicki et al. 1994 trout oral NBT ≠ A. salmonicida ≠

phagocytosis ≠Ž .Anderson et al. 1995 trout ip NBT ≠

im killing ≠Ž .EF203 Yoshida et al. 1993 trout oral phagocytosis ≠ Streptococcus sp. ≠

CL ≠Ž .Sakai et al. 1995f trout oral phagocytosis ≠ R. salmoninarum ™

Ž .BKD vaccine adjuvant NBT ≠

antibody ™Ž .Soybean Rumsey et al. 1994 trout oral NBT ≠

protein phagocytosis ≠

killing ≠Ž .PS-K Park and Jeong 1996 tilapia oral phagocytosis ≠ E. tarda ≠

protein-boundpolysaccharide

Ž .Oligosaccharide Yoshida et al. 1995 catfish oral NBT ≠Ž .Spiralina Duncan and Klesius 1996b catfish oral CL ≠ E. ictaluri ™

antibody ™

()

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Ž .Table 2 continued


Ž .Yeast glucan Robertsen et al. 1990 salmon ip V. anguillarum ≠

V. salmonicida ≠

Y. ruckeri ≠Ž .Raa et al. 1992 salmon oral V. anguillarum ≠

V. salmonicida ≠Ž .Engstad et al. 1992 salmon ip complement ≠

lysozyme ≠Ž .Chen and Ainsworth 1992 catfish ip phagocytosis ≠ E. ictaluri ≠

killing ≠

antibody ≠Ž .Jørgensen et al. 1993a trout ip lysozyme ≠

killing ≠yO ≠2

Ž .Jørgensen et al. 1993b trout ip NBT ™

killing ≠Ž .Rørstad et al. 1993 salmon ip A. salmonicida ≠

Ž .adjuvantA. salmonicida ™

Ž .Sung et al. 1994 shrimp im phenoloxidase ≠ V. Õulnificus ≠

lysozyme ™Ž .Song and Hsieh 1994 shrimp in vitro NBT ≠

CL ≠Ž .Aakre et al. 1994 salmon ip antibody ≠ A. salmonicida ™

Ž .adjuvantŽ .Thompson et al. 1995 trout ip lysozyme ≠ V. anguillarum ™

Ž .Yoshida et al. 1995 catfish oral NBT ≠Ž .Jørgensen and Robertsen 1995 salmon in vitro NBT ≠

killing ™

()

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Ž .Baulny et al. 1996 turbot oral lysozyme ≠ V. anguillarum ≠Ž .adjuvant

complement ™

CL ≠ V. anguillarum ™Ž .Duncan and Klesius 1996a catfish oral CL ≠ E. ictaluri ™

migration ≠Ž .yeast glucan Verlhac et al. 1996 trout oral CL ≠

qVitamin C complement ≠

lysozyme ™Ž .VitaSim Nikl et al. 1991 coho ip A. salmonicida ≠

oral A. salmonicida ≠Ž .Nikl et al. 1993 chinook oral A. salmonicida ≠

im A. salmonicida ™Ž .Ainsworth et al. 1994 catfish oral antibody ≠ E. ictaluri ™

Ž .Peptidoglucan Matsuo and Miyazano 1993 trout oral V. anguillarum ≠Ž .Boonyaratpalin et al. 1995 shrimp oral Yellow-head baculovirus ≠

Ž .Itami et al. 1996 yellowtail oral phagocytosis ≠ E. seriolocida ≠Ž .Lentinan Yano et al. 1989 carp ip phagocytosis ≠ E. tarda ≠

SchizophyllanŽ .Scleroglucan Yano et al. 1991 carp ip A. hydrophila ≠

Ž .Schizophyllan Matsuyama et al. 1992 yellowtail ip complement ≠ E. seriolocida ≠

Scleroglucan phagocytic index ≠ P. piscicida ™

lysozyme ≠Ž .Schizophyllan Itami et al. 1994 prawn oral phagocytosis ≠ Vibrio sp. ≠

Ž .polyglucose Sveinbjornsson and Seljelid 1994 salmon in vitro NBT ≠

pinocytosis ≠

acid phosphatase ≠Ž . Ž .glucan Sigma Jeney and Anderson 1993b trout im phagocytosis ≠

ip NBT ≠


. Ž .1993 . Sakai et al. 1995f reported that R. salmoninarum-vaccinated rainbow troutreceiving EF203 showed higher phagocytic activities and NBT responses in kidneyleucocytes when compared to vaccinated fish without EF203 treatment or to unvacci-nated fish. However, the serum agglutinating antibody titers of vaccinated fish did notshow a significant increase between the EF203 and control groups, and the vaccinatedfish treated with EF203 showed slightly increased survival in comparison with the othergroups following R. salmoninarum challenge.

3.2.8. GlucanŽ .The immune stimulatory effects of glucan have been well-studied Table 2 . The

Ž .effects of several types of glucan; e.g. yeast glucan, peptide-glucan b-1,3, glucan VST ,have been investigated in fish. Yeast glucan is the most extensively studied of these

Ž .glucans. Intraperitoneal injection of yeast glucan b-1,3- and b-1,6-linked glucanprepared from cell walls of Saccharomyces cereÕisiae into Atlantic salmon resulted in

Žincreased resistance to V. anguillarum, V. salmonicida and Y. ruckeri Robertsen et al.,. Ž .1990 . Chen and Ainsworth 1992 reported that catfish injected with yeast glucan

Ž .showed increased resistance to E. ictaluri. However, Thompson et al. 1995 reportedthat rainbow trout injected with yeast glucan did not show enhanced protection againstV. anguillarum infection. Yeast glucan has been applied by immersion and oral

Ž .administration methods. Raa et al. 1992 reported that oral administration of yeastglucan to Atlantic salmon increased protection against V. anguillarum and V. salmoni-

Ž .cida. Tiger shrimp immersed in yeast glucan solution 0.5 and 1 mgrml showedŽ .enhanced protection against V. Õulnificus infection Sung et al., 1994 . Although channel

Žcatfish injected with yeast glucan showed increased protection to E. ictaluri Chen and. ŽAinsworth, 1992 , oral administration did not show such an effect Ainsworth et al.,

.1994; Duncan and Klesius, 1996a .The adjuvant effects of yeast glucan have also been demonstrated. Injection of A.

salmonicida vaccine and yeast glucan into Atlantic salmon enhanced antibody responses

Notes to Table 2:≠: increase.™: no change.trout: rainbow trout.salmon: Atlantic salmon.coho: coho salmon.brook: brook trout.prawn: kuruma prawn.shrimp: black tiger shrimp.chinook: chinook salmon.ip: intraperitoneal injection.im: immersion.NBT: nitroblue tetrazolium reaction.CL: chemiluminescent response.IL1: interleukin 1 production.MAF: macrophage activating factor.killing: bactericidal activity of macrophage.Oy: production of superoxide anion.2


Ž .Aakre et al., 1994 and induced significantly increased protection against furunculosisŽ .over vaccines without yeast glucan Rørstad et al., 1993 . The injection of yeast glucan

Ž .alone did not show protection. Baulny et al. 1996 reported that oral administration ofyeast glucan to turbot immersed in V. anguillarum bacterin also increased protectioncompared with bacterin alone. As in the experiment above, yeast glucan alone did notenhance protection against V. anguillarum infection.

Yeast glucan enhances the lysozyme activity in Atlantic salmon, rainbow trout andŽturbot Engstad et al., 1992; Jørgensen et al., 1993a; Thompson et al., 1995; Baulny et

. Ž .al., 1996 , complement activity Engstad et al., 1992 , bacterial killing activity ofŽmacrophages in rainbow trout, Atlantic salmon and catfish Chen and Ainsworth, 1992;

.Jørgensen et al., 1993a,b; Jørgensen and Robertsen, 1995 and the production ofsuperoxide by macrophages or hemocytes in rainbow trout, tiger shrimp, catfish and

ŽAtlantic salmon Jørgensen et al., 1993a; Song and Hsieh, 1994; Yoshida et al., 1995;.Baulny et al., 1996 .

Ž .b-1, 3 glucan VST derived from Schizophyllum commune has also been evaluatedas a protection against disease. Coho salmon treated with VST by injection or oral routes

Ž .showed enhanced protection against A. salmonicida Nikl et al., 1991, 1993 . However,Žchinook salmon immersed in a solution of this glucan did not show protection Nikl et

. Ž .al., 1993 . Ainsworth et al. 1994 reported that catfish fed VST did not show increasedserum lysozyme activity, although they showed increased production of superoxideanion by macrophages and reduced mortality when challenged with E. ictaluri. Further-more, this glucan showed no adjuvant effect on E. ictaluri bacterins despite increased

Ž .antibody titers Ainsworth et al., 1994 .Peptidoglucan prepared from BreÕibacterium lactofermentum increased phagocytosis

Ž .in yellowtail and resistance to Enterococcus seriola infection Itami et al., 1996 . Theefficacy of peptidoglucan was also demonstrated against vibriosis in rainbow troutŽ .Matsuo and Miyazano, 1993 and yellow-head baculovirus infection in black tiger

Ž . Žshrimp Boonyaratpalin et al., 1995 . The efficacies of glucan supplied by Sigma Jeney. Žand Anderson, 1993b , Lentinan, Schizophyllan and Scleoglucan Yano et al., 1989,

.1991; Matsuyama et al., 1992; Itami et al., 1994 have also been demonstrated inrainbow trout, carp, yellowtail and prawn.

3.3. Animals and plant components

3.3.1. Animal extractsThe extracts from some invertebrates have immunostimulatory effects. An extract

Ž .from the marine tunicate, Ecteinascida turbinata Ete and a glucoprotein fraction ofŽ .water extract Hde from abalone, Haliotis discus hannai, enhanced the killing of tumor

Ž .cells in vitro and inhibited tumor growth in vivo Sigel et al., 1970; Uchida et al., 1987 .Eel injected with Ete showed enhanced phagocytosis and increased survival following

Ž . Ž .A. hydrophila challenge Davis and Hayasaka, 1984 . However, Stanley et al. 1995demonstrated that the survival after infection with E. ictaluri decreased in channelcatfish injected with Ete, although immunoenhancement was observed. Rainbow troutinjected with Hde also showed enhanced phagocytic and NK cell activities, and showed

Ž .increased survival against V. anguillarum infection Sakai et al., 1991 . The heat extract


from firefly squid, Watasenia scintillans, also stimulated the immune system of rainbowtrout such as the production of superoxide anion, potential killing activities by

Žmacrophages and the lymphoblastic transformation of lymphocytes in vitro Table.Ž .3 Siwicki et al., 1994 .

3.3.2. GlycyrrhizinGlycyrrhizin is a glycosylated saponin, containing one molecule of glycyrretinic acid,

which has anti-inflammatory and anti-tumor activities, mediated by its immunomodula-Ž . Ž .tory activities Wada et al., 1987; Zhang et al., 1990 . Edahiro et al. 1990, 1991

reported that yellowtail treated orally with glycyrrhizin showed increased protectionagainst E. seriola infection, although lysozyme activity of blood and phagocytic

Ž .activities of macrophages were not enhanced. However, Jang et al. 1995 reported thatin vitro treatment with glycyrrhizine enhanced the respiratory burst activity ofmacrophages and the proliferative responses of lymphocytes from rainbow trout.

3.3.3. Other immunostimulantsOther immunostimulants are also shown in Table 2. Rainbow trout treated orally with

soybean protein showed increased leucocyte activities such as phagocytosis, bacterialŽ .killing and the production of superoxide Rumsey et al., 1994 . The bath administration

of Quil A saponin with Y. ruckeri vaccine enhanced the in vitro bactericidal activities inŽ . Ž .rainbow trout Grayson et al., 1987 . Ninomiya et al. 1995 reported that oral

administration of Quil A saponin increased leucocyte migration in yellowtail. TheŽ .immunostimulant effects of PS-K protein-bound polysaccharide , Spiralina, acid pep-

tide fractions from fish protein hydrolysate were also reported in tilapia, OreochrmisŽ . Ž .niloticus, by Park and Jeong 1996 , channel catfish by Duncan and Klesius 1996b and

Ž .Atlantic salmon by Gildberg et al. 1996 , respectively.

3.4. Diet components

Dietary vitamin C is essential for normal growth and for several physiologicalŽ .functions in most fishes Halver, 1989 . High levels of dietary vitamin C are reported to

increase resistance to Edwardsiella tarda and E. ictaluri infection in channel catfishŽ .Durve and Lovell, 1982; Li and Lovell, 1985; Liu et al., 1989 , to V. anguillarum, IHN

Žand Ichthyophthirius multifiliis in rainbow trout Navarre and Halver, 1989; Suzuki and.Ai, 1989; Wahli et al., 1995 and to A. salmonicida and V. salmonicida in Atlantic

Ž . Ž .salmon Erdal et al., 1991; Hardie et al., 1991 . Li and Lovell 1985 and Hardie et al.Ž .1991 showed that treatment with high doses of vitamin C increased complement

Ž .activities in catfish or Atlantic salmon. However, Yano et al. 1988 and Liu et al.Ž .1989 did not observe such effects in catfish or red sea bream, respectively. On the

Ž .other hand, the activation of macrophages was reported by Thompson et al. 1993 inŽ . Ž .Atlantic salmon and by Roberts et al. 1995 in turbot. Hardie et al. 1991 reported that

treatment of fish with high doses of vitamin C stimulated macrophage activating factors,Ž .followed by lymphocyte proliferation Hardie et al., 1993 . These results show that

Ž .fishes fed with high doses more than 1000 mgrkg of vitamin C have protectiveŽ .immune responses Table 4 .

()

M.Sakair

Aquaculture

1721999

63–

9277

Table 3Animal and plant extracts used as fish immunostimulants


Ž . Ž .Tunicate Ete Davis and Hayasaka 1984 eel ip phagocytosis ≠ A. hydrophila ≠Ž .Stanley et al. 1995 catfish ip antibody ™ E. ictaluri x

phagocytosis ≠Ž . Ž .Abalone Hde Sakai et al. 1992 trout ip CL ≠ V. anguillarum ≠

phagocytosis ≠

NK ≠Ž .Firefly squid Siwicki et al. 1996 trout ip NBT ≠

Ž .mitogen Con A, LPS ≠

killing ≠Ž .Acid peptide fractions from Gildberg et al. 1996 salmon in vitro NBT ≠

fish protein hydrolysateŽ .Quil A saponin Ninomiya et al. 1995 yellowtail oral leucocyte migration ≠

Ž .Grayson et al. 1987 trout im serum bactericidal activity ≠Ž .Glycyrrhizin Edahiro et al. 1990 yellowtail oral complement ≠ E. seriolicida ≠Ž .Edahiro et al. 1991

Ž . Ž .Jang et al. 1995 trout in vitro mitogen Con A, LPS ≠

MAF ≠yO ≠2

≠: increase.™: no change.x: decrease.trout: rainbow trout.salmon: Atlantic salmon.CL: chemiluminescent response.NK: natural killer cell.NBT: nitroblue tetrazolium reaction.Con A: concanavalin A.MAF: macrophage activating factor.ip: intraperitoneal injection.im: immersion.


Table 4Immunostimulant effects of diet components in fish

Authors Doses Fish Results Resistance toŽ .mgrkg pathogens

Vitamin CŽ .Durve and Lovell 1982 150 catfish E. tarda ≠

14 wŽ .Li and Lovell 1985 3000 catfish complement ≠ E. tarda ≠

antibody ™

phagocytic index ≠

macrophage killing ™Ž .Liu et al. 1989 4000 catfish complement ™ E. tarda ≠

9 w antibody ™Ž .Yano et al. 1988 1000 Red sea complement ™

breamŽ .Suzuki and Ai 1989 550 trout IHNV ≠

10 dŽ .Navarre and Halver 1989 2000 trout V. anguillarum ≠

12 wŽ .Erdal et al. 1991 4000 salmon V. salmonicida ≠

52 dŽ .Hardie et al. 1991 2750 salmon complement ≠ A. salmonicida ≠

26w NBT ™

phagocytosis ™

MAF ≠Ž .Johnson and Ainsworth 1991 1000 catfish neutrophil ™

Ž .7 w phagocytosisŽ . Ž .Hardie et al. 1993 in vitro 244 trout proliferation ≠

16 w NBT ™

MAF ≠Ž .Thompson et al. 1993 3170 salmon NBT ≠

16 w killingŽ .A. salmonicida ≠

migration ≠

antibodyŽ .A. salmonicida ≠

Ž .Roberts et al. 1995 2000 turbot phagocytic index ≠

127 d lysozyme ≠™Ž .Wahli et al. 1995 2000 trout I. multifiliis ≠

30 d

Vitamin EŽ .Blazer and Wolke 1984 low levels trout antibody x

Ž .Hardie et al. 1990 800 salmon NBT ™ A. salmonicida ≠

20 w complement ≠

lysozyme xŽ .Wise et al. 1993a 240 catfish NBT ≠

120 dSe 0.8

Ž .Wise et al. 1993b 2500 catfish phagocytic index ≠

180 d antibody ™


Ž .Table 4 continued

Authors Doses Fish Results Resistance toŽ .mgrkg pathogens

Vitamin EŽ .Pulsford et al. 1995 500 turbot phagocytosis ≠

12 wŽ .Taksdal et al. 1995 low levels salmon IHNV x

Vitamin AŽ .Thompson et al. 1994 salmon anti-protease activity ≠ A. salmonicida ≠

migration ≠

w: week.d: day.≠: increase.™: no change.x: decrease.trout: rainbow trout.salmon: Atlantic salmon.NBT: nitroblue tetrazolium reaction.MAF: macrophage activating factor.Se: selenium.

ŽVitamin E also enhances both humoral and cellular defenses in mammals Panush. Ž .and Delafuente, 1985 . Blazer and Wolke 1984 reported that specific and cell-mediated

immunity and macrophage phagocytosis were all compromised in rainbow trout fedŽ .vitamin E-depleted diets. Furthermore, Hardie et al. 1990 reported that Atlantic salmon

fed vitamin E-depleted diets had significantly increased mortality rate following A.salmonicida infection compared to fish on the commercial diet. However, only comple-

Ž .ment activity was compromised in these vitamin E-depleted fish. Wise et al. 1993a,bshowed that catfish fed high doses of vitamin E had an increased phagocytic indices and

Ž .superoxide anion production by leucocytes Table 4 .Other dietary components such as lipids, other vitamins, trace elements, etc. can

affect immune responses in fish. The importance of diet in fish immune responses wasŽ . Ž .reviewed by Landolt 1989 and Blazer 1992 .

3.5. Hormones, cytokines and others

3.5.1. HormonesThe relationship between neuroendocrine regulation and the immune system has

Žrecently become the subject of intense investigation Berczi and Nagy, 1987; Kelley,.1989; Gala, 1991; Auernhammer and Strasburger, 1995 . It is also known that GH and

Ž .PRL directly affect immunocompetent cells macrophages, lymphocytes and NK cellsŽ . Ž .Table 5 . In fish, exogenous growth hormone GH has mitogenic activity on lympho-

Ž .cytes and activates NK cells Kajita et al., 1992b; Sakai et al., 1996b . In addition, SakaiŽ .et al. 1995d, 1996a reported that exogenous GH given to rainbow trout increased the

Ž .production of superoxide anion in leucocytes. Calduch-Giner et al. 1995 detected GHreceptors in monocytes and lymphocytes of gilthead sea bream, Sparus aurata. Re-


Table 5Hormones and cytokines used as fish immunostimulants


Ž .LF Sakai et al. 1993 trout oral phagocytosis ≠ V. anguillarum ≠

CL ≠ Streptococcus sp. ™Ž .Sakai et al. 1995b trout in vitro CL ≠

NBT ≠Ž .Kakuta et al. 1996 red sea oral lectin ≠ Cryptocaryon ≠

bream lysozyme ™Ž .GH Kajita et al. 1992b ip NK ≠Ž .Sakai et al. 1995d trout ip CL ≠Ž .Sakai et al. 1996a trout in vitro NBT ≠

mitogen ≠Ž .Sakai et al. 1997 trout ip phagocytosis ≠ V. anguillarum ≠Ž .PRL Sakai et al. 1996a trout in vitro NBT ≠Ž .INF Tamai et al. 1993 hirame in vitro HRV ≠

LF: lactoferrin.GH: growth hormone.PRL: prolactin.INF: interferon.trout: rainbow trout.≠: increase.™: no change.CL: chemiluminescent response.NBT: nitroblue tetrazolium reaction.NK: natural killer cell.ip: intraperitoneal injection.

Ž .cently, Sakai et al. 1996c reported that injection of rainbow trout with GH increasedserum hemolytic activities. Furthermore, the injection of GH increased protection

Ž .against V. anguillarum in rainbow trout Sakai et al., 1997 .Ž . Ž .Prolactin PRL also has immunostimulatory activities. Sakai et al. 1996b reported

that the addition of homogeneous PRL to chum salmon, Oncorhynchus keta, inducedlymphocyte mitogenic responses and PRL increased the production of superoxide anion

Ž . Ž .by leucocytes in rainbow trout Sakai et al., 1996a . Sakai et al. unpublished alsoreported that b-endorphin activated the phagocytic activity of rainbow trout leucocytes.

3.5.2. CytokinesCytokines are polypeptides or glycoproteins which act as modulators in the immune

Žsystem. The existence of several cytokines has been reported in fish Secombes et al.,.1996 . The structures of cytokines such as interleukin 2 have already been reported

Ž .Tamai et al., 1992 . Several cytokines can be used as immunostimulants. Tamai et al.Ž .1993 reported that a recombinant interferon-like protein produced from Japanese

Žflatfish, Paralichthys liÕaceus, tha amino acid homology was less than 20%, with.murine interferon a or b molecule , inhibited infection of EPC cells by hirame

rhabdovirus. Although the use of cytokines as immunostimulants has only been de-Ž .scribed in one report by Tamai et al. 1993 , cytokines may be useful as powerful


immunostimulants if their structures can be identified and recombinant moleculesprepared.

3.5.3. LactoferrinLactoferrin, which consists of a single peptide chain with a molecular weight of about

87,000 and possessing two Fe-binding sites per molecule, is widely distributed in thephysiological fluids of mammals. Many biological functions have been attributed to

Žlactoferrin including antibacterial activity Arnold and Brewer, 1980; Bortner et al.,. Ž .1986 , regulation of Fe absorption Kawakami et al., 1988 and growth promotion of

Ž .macrophages Amberuso and Johnston, 1981 . It has also been found to regulatehydroxyl radical production by macrophages, granulocytes and neutrophil leucocytesŽ . Ž .Amberuso and Johnston, 1981; Gahr et al., 1991 . Sakai et al. 1993 reported thatrainbow trout orally treated with bovine lactoferrin showed enhanced phagocytic activi-ties and the production of superoxide anion by macrophages, and demonstrated high

Ž .resistance 40-fold increase in LD to V. anguillarum infection but marginal increase50Ž . Ž .in resistance 4-fold increase in LD to Streptococcus sp. Sakai et al., 1993 .50

Ž .Lactoferrin can activate macrophages in vitro Sakai et al., 1995b . Recently, KakutaŽ . Ž .and Kurokura 1995 and Kakuta et al. 1996 reported that red sea bream, P. major,

orally administered bovine lactoferrin showed increased numbers of granulocytes andlymphocytes in the blood, elevated secretion of body mucus and resistance to Crypto-caryon irritans infection.

4. Therapeutic regiments for immunostimulant application

The use of immunostimulants can protect fish from several infectious diseases andŽ .decrease mortality rates Table 6 ; however, fish cannot be protected against all

infectious diseases by immunostimulants. Fishes receiving immunostimulants showincreased resistance against infection by bacteria such as V. anguillarum, V. salmoni-cida, A. salmonicida, and Streptococcus sp., viral infection such as IHN and yellow-headbaculovirus, and parasite infection such as white spot disease. Positive effects ofimmunostimulants against many virus infections such as VHS, IHN, etc. and otherbacterial infections such as Cytophaga infections have not been reported.

Immunostimulants do not increase resistance against R. salmoninarum, P. piscicidaŽ .or E. ictaluri infection Table 7 . These bacteria are resistant to phagocytosis and can

Žsurvive within macrophages Nelson et al., 1989; Baldwin and Newton, 1996; Guten-.berger et al., 1997 . As already indicated, the main immunological function increased by

immunostimulants is the activity of phagocytic cells. However, macrophage-resistantbacteria may escape from activated macrophages and thus in these situations, immunos-timulants do not appear effective against such infections.

5. Timing of immunostimulants administration

The effect of timing the administration on immunostimulant function is a veryimportant issue. Usually, the most effective timing of antibiotics is upon the occurrence


Table 6Effectiveness of the use of immunostimulants on infectious diseases

EffectiÕeBacterial infections

Ž .V. Õulnificus yeast glucanŽV. anguillarum yeast glucan, lactoferin, HDe, levamisole, chitin, Vitamin C,

.C. butyricum cells GH, MDP, PGŽ .V. salmonicida yeast glucan, Vitamin C, Vitamin EŽ .A. salmonicida Vitamin C, FK-565, yeast glucan, A. stenohalis cells, chitosan, levamisole, VSTŽ .A. hydrophila levamisole, Ete

Ž .Y. ruckeri yeast glucanŽ .Streptococcus sp. EF203

Ž .E. seriolocida Schizophyllan, Scleroglucan, Glycyrrhizin, PGŽ .E. ictaluri yeast glucan

Ž .E. tarda Vitamin C, PS-K, Lentinan, Schizophyllan, ScleroglucanViral infections

Ž .IHN Vitamin CŽ .Yellow-head baculovirus yeast glucan

Parasite infectionsŽ .I. multifiliis Vitamin C

Ž .Cryptocaryon sp. lactoferrin

Non-effectiÕeBacterial infections

Ž .A. salmonicida yeast glucanŽ .V. anguillarum yeast glucan

Ž .E. ictaluri Ete, yeast glucan, VST, SpiralinaŽ .P. piscicida Schizophyllan, Scleroglucan, FCA, yeast glucan, chitin

Ž .R. salmoninarum EF203

of disease, and they cannot often be used prophylactically due to risk of fostering theŽ .development of drug-resistant bacteria. Anderson 1992 proposed that immunostimu-

lants should be applied before the outbreak of disease to reduce disease-related losses.Immunostimulants can promote recovery from immunosuppression states caused by

Ž .stress. Kitao and Yoshida 1986 reported that rainbow trout injected with cyclophospha-mide or hydrocortisone showed suppressed phagocytic activity of peritoneal and kidneyleucocytes, and this suppression of the phagocytosis was reversed by injection of

Ž .FK-565. Boonyaratpalin et al. 1995 also showed that peptidoglycan-fed black tigershrimp exhibited a higher tolerance to dissolved oxygen, salinity, and stress than thosefed the control diet.

Table 7A comparison of characteristics of chemotherapeutics, vaccines and Immunostimulants

Chemotherapeutics Vaccine Immunostimulants

When Therapeutically Prophylactically ProphylacticallyEfficacy Excellent Excellent GoodSpectrum of activity Middle Limited WideDuration Short Long Short


The effective timing of administration of immunostimulants such as Ete is verydifficult. Ete exerted a protective effect in eels injected intraperitoneally 2 days afterchallenge with A. hydrophila. However, the protection was not seen when Ete was

Žadministered intraperitoneally 2 days before or concurrently with the bacteria Davis and.Hayasaka, 1984 .

6. Route of administration

Many authors reported that the injection of immunostimulants enhances the functionof leucocytes and protection against pathogens. However, this method is labor intensive,relatively time-consuming and becomes impractical when fishes weigh less than 15 g.Thus, another method such as oral administration or immersion should be used. Oraladministration of immunostimulants has already been reported for glucans, EF203,lactoferrin, levamisole and chitosan. This method is non-stressful and allows massadministration regardless of fish size. Oral administration of these immunostimulantsresults in enhancement of leucocyte function and protection against infectious diseasessuch as furunculosis, vibriosis, streptococcosis. The immersion method can also used.

Ž . ŽBaba et al. 1993 reported that carp immersed in levamisole solution 10 mgrml for 24.h showed activated phagocytic activities, chemotactic abilities and the production of

active oxygen in head kidney phagocytes and enhanced protection against A. hy-Ž .drophila. This effect lasted for at least 2 weeks. Anderson et al. 1995 demonstrated

that rainbow trout immersed with glucan or chitosan showed increased protectionagainst A. salmonicida after treatment for 3 days. However, this effect was transient andwas not present after 14 days. Adjuvant effects of immunostimulants administered by

Ž .immersion have also been reported by several authors. Jeney and Anderson 1993areported that rainbow trout bathed in A. salmonicida O-antigen in combination with

Žimmunostimulants levamisole, quarternary ammonium compound, polypeptide of fish. Ž .extract 30 min immersion enhanced phagocytosis by leucocytes and antibody titers

Ž .against A. salmonicida, and showed adjuvant effects with vaccination. Nikl et al. 1993Žcompared the adjuvant effects of glucan against A. salmonicida vaccine oral delivery 7

. Ž .days administration and immersion 15 min . No adjuvant effects were seen with theimmersion treatment, although the fish administered glucan orally showed enhancedvaccine effects. The efficacies of immersion administration of immunostimulants hasbeen demonstrated by several authors. However, the dilution and the levels of efficacyrequire more complete investigation.

7. Doses

Immunostimulants increase the immune responses and enhance protection againstŽ .pathogens which raises the question of dose-dependency. Kajita et al. 1990 showed

that the chemiluminescent effects of phagocytic cells in rainbow trout were increased byinjection of levamisole at 0.1 and 0.5 mgrkg. However, they also reported that theinjection of 5 mgrkg of levamisole did not produce this immunostimulant effect.


Ž .Robertsen et al. 1994 reported that the increase in respiratory burst activity ofglucan-treated macrophages was maximal at glucan concentrations of 0.1–1 mgrml,whereas at 10 mgrml no effect was seen and at 50 mgrml glucan was inhibitory.

Ž .Comparable effects on function were also observed in lymphocytes. Kitao et al. 1987Ž .reported that high doses 10 mgrkg of FK 565 did not increase the numbers of

Ž . Ž .plaque-forming cells PFC against Y. rukeri, although the optimum dose 5 mgrkgincreased PFC. The effects of immunostimulant are therefore not directly dose-depen-dent, and high doses may not enhance and may inhibit the immune responses.

8. The effects of long-term administration

Oral administration is the most practical method for delivery of immunostimulants;however, the effects of long-term oral administration of immunostimulants are still

Ž .unclear. Matsuo and Miyazano 1993 reported that rainbow trout treated with pepti-doglucan orally for 56 days did not show protection after challenge with V. anguillarum,

Ž .although fish treated for 28 days showed increased protection. Yoshida et al. 1995 alsodemonstrated that the number of NBT-positive cells in African catfish increasedfollowing oral administration of glucan or oligosaccharide over 30 days, but not over 45days. Although the reasons for these decreases in immune responses in fish by long-termoral administration of immunostimulants is still unknown, negative feedback systemsagainst immunostimulation may function in fish, and the immune response may revert toa previous state. Thus, the effective administration period should be investigated foreach immunostimulant.

9. The additional effects of immunostimulants

Several authors have reported relationships between immunostimulation and growth-Ž .promoting activity. Boonyaratpalin et al. 1995 reported that black tiger shrimp fed with

peptidoglycan-supplemented feed showed better growth and feed conversion rates thanthose fed a normal diet. This effect was observed with 0.01% peptidoglycan supplemen-

Ž . Ž .tation, but not with the highest level administered 0.1% . Sung et al. 1994 alsoshowed that black tiger shrimp grew faster with glucan immersion at the 0.5, 1, and 2mgrml than at 0.25 mgrml or in control solutions. However, Matsuo and MiyazanoŽ .1993 reported that peptidoglucan did not influence rainbow trout growth after 60 days

Žof oral administration. It is well-known that GH promotes the growth of fish Rand-. Ž .Weaver and Kawauchi, 1993 . Sakai et al. 1995d, 1996a,b showed that GH also

functions as an immunostimulant and enhances macrophage activities of fish. Rainbowtrout injected with GH exhibited increased resistance against V. anguillarum challenge.Thus, there may be a close correlation between growth and immunostimulation.

On the other hand, the mere deleterious side effects of immunostimulants have notbeen investigated. As already described in this review, overdoses and long-term adminis-tration of immunostimulants reduce their efficacy. In aquaculture, the maturation andspawning of fish are very important processes. No research has yet been performed


concerning the influence of immunostimulants at these stages. When fish spawn, theŽ .immune systems become suppressed by sex hormones Wang and Belosevic, 1995 .

Although the use of immunostimulants could cause recovery of the immune systemssuppressed by sex hormones, they may disturb sexual maturation and other essentialfunctions associated with spawning.

10. Conclusions

In this review, the benefits of immunostimulants for aquaculture were discussed. Thecharacteristics of immunostimulants are shown in Table 7. There are mainly threemethods for control of fish disease: vaccination, chemotheraputics and immunostimu-lants. Both immunostimulants and vaccines are used to prevent infectious diseases.However, we cannot expect the marked or long-term effects observed with vaccines tooccur with immunostimulants. Although immunostimulants may be used for treatment ofsome infectious diseases, they are not as effective as many chemotheraputics. Immunos-timulants are, however, not inferior to vaccines and chemotherapeutics. Antibiotic-re-sistant bacteria can prevail in the aquaculture environment and the treatment of fishdisease using chemotherapeutics has thus become more difficult. Although vaccinationis the most reliable method to control fish diseases, there are, as yet, no effectivevaccines against BKD or most viral infections. Immunostimulants may be able tocompensate for these limitations of chemotherapeutics and vaccines. Immunostimulantsare thought to be safer than chemotheraputics and their range of efficacy is wider thanvaccination. The combination of vaccination and immunostimulant administration mayalso increase the potency of vaccines. Thus, with a detailed understanding of theefficacy and limitations of immunostimulants, they may become powerful tools tocontrol fish diseases.

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sakai(1999)

Documents

mature fish

fish andshrimp

antibodyresponses of

infectious diseases

health of cultured fish

control of fish diseases

fish culture aoki

effects of immunostimulants