lournal of Fish Diseases 1990, 13, 391-400

Enhancement of non-speeifie disease resistanee inAtlantie salmon, Salmo salar L,, by a gluean fromSaccharomyces cerevisiae eell walls

B. ROBERTSEN,^ G. R0RSTAD,^ R. ENGSTAD^ & J. RAA^ Departments of'PlantPhysiology and Microbiology, IBG, and '^Marine Biochemistry, Norwegian College of Fishery Science, University

of Troms0, and '^Center of Aquaculture Research, Troms0, Norway

Abstract, An insoluble polysaccharide from the cell wall of Saccharomyces cerevisiae, calledM-Glucan, has been shown to enhance the non-specific disease resistance of Atlanticsalmon, Salmo salar L., when injected intraperitoneally. M-Glucan consists only of glucoseunits which presumably are linked through j6-l,3 and /5-l,6 linkages. Enhanced resistancewas demonstrated against Yersinia ruckeri, the causal agent of enteric redmouth disease,against Vibrio anguitlarum., the causal agent of classical vibriosis and against Vibriosalmonicida, which causes cold water vibriosis or 'Hitra-disease' in salmon. At a dose of2mg M-Glucan per fish (20g mean weight), maximal resistance developed in the fish3 weeks after injection. Injection of different gluean doses and challenge one week laterwith Vibrio anguillarum, showed that 50-200/^g gluean per fish resulted in the highest levelof resistance. The level of resistance in Atlantic salmon obtained with M-Glucanwas strikingly higher than that obtained with another gluean which was prepared fromSaccharomyces cerevisiae by a different procedure.

Introduction

The use of vaccines and antibiotics are the two available direct methods to protect farmed fishagainst diseases. Vaccines with a high degree of efficacy have been developed against somebacterial diseases, such as the 'Hitra-disease' in Atlantic salmon, Salmo salar L., (Holm &J0rgensen 1987). However, no efficient vaccine exists yet against such commercially importantdiseases as furunculosis and bacterial kidney disease in salmonids, and against viral diseases. Insome cases, the pathogenic agent is unknown and therefore beyond the scope of a classicalapproach to vaccine development. This is the case with the infectious anaemia disease ofsalmon which is an increasing problem in Norwegian fish farms.

The use of antibiotics has to be considerably reduced in aquaculture, primarily to avoidenvironmental hazards and the spread of antibiotic resistance genes. In addition, such com-monly used antibiotics as the oxytetracyclines also suppress immune responses in fish (Rijkers,van Oosterom & van Muiswinkei 1981).

Unlike vaccines (=antigens) which trigger the production of specific antibodies towards oneparticular pathogen, a group of biological and synthetic compounds exists which enhance thenon-specific defence mechanisms in animals (Fenichel & Chirigos 1984). Such compounds mayrepresent an alternative to vaccines and antibiotics in the protection of farmed fish againstdiseases.

Killed mycobacteria and muramyl dipeptide have been reported to enhance resistance ofrainbow trout, Oncorhynchus mykiss (Richardson), against several different bacterial pathogens

Correspondence: B0rre Robertsen, Department of Plant Physiology and Microbiology, IBG, University ofTroms0, N-9(KJ0 Troms0, Norway.

391

392 R. Robcttsvn ct al.

(Olivier, Evelyn &. Lallicr 1985). Kitao & Yoshida (1986) reported that the synthetic peptideFR-565 enhanced the non-specific resistance of rainbow trout against Aeromonas salmonicida.Furlhcrniorc, an extract from a marine tunicate has been reported to increase resistance in theAmerican ccl, Anguilla rostrata Lc Sucur, against infection by Aeromonas hydrophila (DavisLK: Hayasaka U)84).

Elements of the non-specific defence mechanisms in vertebrates include the phagocyticcells, neutrophils and macrophages, and complement, lysozyme, C-reactive protein, interferonand transferrin (Fletcher 1982; Lamcrs 1985). Although little is known about the mechanism ofaction of immunostimulatory compounds in fish, some of them appear to enhance the microbekilling activity of the macrophages (Olivier, Eaton & Campbell 1986).

The present authors' research group has initiated investigations on the potential use ofyeast cell wall glucans to enhance the non-specific defence mechanisms in fish. These areinsoluble polysaceharides composed of glucose units which are linked through /3-l,3 and ^1,6linkages (Duffus, Levi & Manners 1982). Glucans are one of the most important structuralelements of fungal cell walls in general (Rosenberger 1976), and are known to stimulatedefence mechanisms in a range of higher organisms. In plants, they stimulate production oflow molecular weight antibiotics called phytoalexins (Darvill & Albersheim 1984); in invert-ebrates, glucans have been shown to activate polyphenoloxidase, a defence enzyme in thehaemolymph (Unestam & Soderhall 1977); and in mice, glucans have been shown to enhanceanticancer mechanisms as well as the non-specific defence against microbiai pathogens (reviewedby DiLuzio 1985). DiLuzio and co-workers have documented that a cell wall glucan fromSaccharomyces cerevisiae has the ability to enhance the resistance in mice against bacterial,fungal and viral pathogens (Di Luzio, Williams, McNamee, Edwards & Kitahama 1979;DiLuzio 1983; Williams, Browder & DiLuzio 1983).

The authors' group has, in cooperation with the biotechnology company Mackzymal AS,developed a glucan preparation from S. cerevisiae cell walls, called M-Glucan, which has theability to enhance non-specific disease resistance in the Atlantic salmon, as described in thepresent paper.

Materials and methods

Chemicals

M-Glucan is an insoluble polysaccharide which was prepared from the cell walls of S. cerevisiaeby Mackzymal AS, Troms0, Norway. The glucan was suspended in 0-9% NaCI by ultra-sonication using a Branson B-12 sonicator equipped with a microtip probe. DL-Glucan is aninsoluble glucan which was prepared from S. cerevisiae according to the procedure describedby DiLuzio et al. (1979). Cellulose (Sigmacell, Type 20) and ethyl p-aminobenzoate wereobtained from Sigma.

Bacteria

Bacteria were stored in 20% glycero! at -80*'C.Vibrio salmonicida NCMB 2262 was grown overnight in marine broth medium (Difco) at

I5°C, then washed and resuspended in 1 3 % NaCl in 0-1 M sodium phosphate buffer at pH 7-2for the challenge experiments.

Yersinia ruckeri serovar 01 and Vibrio anguillarum serovar 01 were provided by

Fnhanccment of resistance in salmon by M-Glucan 393

Apothckerncs laboratorium AS, rroms0. The bacteria were grown for about 20h at I5°C inbrain heart infusion broth (Difco) and then washed and resuspended in 0-85% NaCl in 0-1 Msodium phosphate buffer at pH 7 2 for the challenge experiments.

Fish and challenge experiments

Challenge experiments with fish were carried out at the veterinary station for contract researchin fish diseases, Vikan, Norway, and at FORUT, Troms0, Norway. Presmolt Atlantic salmonwith an average weight of 20—35 g were obtained from a local smolt producer and kept in 2001flow-through tanks supplied with aerated fresh water. Groups of 30—50 fish were injectedintraperitoneally (i.p.) with a 0 2ml suspension of glucan in saline or with 0-2mI saline as thecontrol. The eontrol fish and the glucan-treated fish were kept in the same tank and werelabelled by fin-clipping. The fish were ehallenged 1—8 weeks later by i.p. injection of a 0-1 mlsuspension of a baeterial pathogen. Prior to injection of gluean, saline or pathogenic bacteria,the fish were anaesthetized with ethyl p-aminobenzoate (0-04mg/ml). The number of dead fishwere recorded daily following inoeulation. In the ehallenge experiments with Y. ruckeri and V.anguillarum, the water temperature was kept at an average of about 12°C unless otherwisestated. In experiments with V. salmonicida, the water temperature was kept at 9—10°C.

Analyses of M-Glucan

The eontent of hexose in M-Glucan was determined after dissolution of the gluean in 67% (v/v) H2SO4 by the anthrone method (Dische 1962) with cellulose as the standard. Monosaecharideeomposition was determined by GLC by the alditol acetate method (Albersheim, Nevins,English & Karr 1967) using an SP-2380 partially crosslinked eyanosilicone capillary column(30m X 0-25 mm i.d., film thickness Q-ljmv) in a Hewlett Packard 5880 A Series GC with aFID detector. The samples were injected with a split ratio of 1:100, the oven temperature waskept at 250°C for 12 min and then raised to 260°C at a rate of 20'^C/min. Nitrogen wasdetermined by the Kjeldahl method on a Kjeltech autoanalyser (Tecator, Sweden).

Results

A summary of the experiments showing enhancement of disease resistance in the Atlanticsalmon with M-Glucan is presented in Table 1. In six different ehallenge experiments withV. salmonicida, the causal agent of cold water vibriosis, i.p. injeetion of M-Gluean in salmonresulted in a relative percentage protection (RPP) of 53-81%. Figure 1 illustrates the devel-opment of mortahty in the glucan-treated and control fish.

Six different experiments showed that M-glucan also increased the resistance in salmonagainst V. anguillarum, the causal agent of classical vibriosis in salmonids (Table 1). The RPPobtained was from 41 to 58%. Figure 2 illustrates the development of mortality in the glucan-treated and control fish inoculated with V. anguillarum.

In experiments 7 and 8, Table 1, the fish were first challenged 4 weeks after injection with8x10-^ and 7x10'* V. anguillarum per fish respectively, but as no mortality occurred the fishwere rechallenged 4 weeks later with 2 - 4 X H / ' and 6x10^^ V. anguillarum per fish, respectively.A relatively high inoculum had to be used because the water temperature (6-9''C) wassuboptimal for the outbreak of vibriosis. In this experiment, the increased resistance observedin the glucan-treated fish may be due to enhanced specific resistance because the unsuccessful

394 B. Robertseti et al.

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Enliaticemcnt of resistance in salmon hy M-Glucan 395

10 15Days after infection

Figure 1. Cumulative mortahty in Atlantic salmon treated with M-Glucan or saline after challenge with Vibriosalmonicida. Atlantic salmon with a mean weight of 20g were injected i.p. with a 0-2ml suspension of 2mgM-Glucan in saline (•) or with 0-2mI sahnc (•). The fish were challenged 3 weeks later by i.p. injection ofSxlO-' bacteria per fish (n=5(i).

challenge may have induced specific immunity towards V. angtitllarum. If that is the case, theprotective effect of M-Glucan is likely to be an adjuvant effect.

Figure 3 shows the effect of M-Glucan on the resistance of salmon against Yersinia ruckeri,the causal agent of enteric redmouth disease. M-Glucan delayed the onset of mortalitycompared to the saline control and also reduced the total mortality of the fish. The RPP was,however, lower in experiments with Y. ruckeri than in the experiments with V. anguillarumand V. salmonicida (Table 1).

In several experiments, we compared the protective effect of M-Glucan with the effectobtained with glucan prepared from S. cerevisiae according to the procedure of DiLuzio et al.(1979). The latter glucan is called DL-Glucan. Although DL-Glucan increased the resistanceof salmon against V. salmonicida., the protective effect of DL-Glucan was much lower thanthat of M-Glucan. This is illustrated in Fig. 4. In this experiment, only 24% of the fish whichhad been treated with DL-Glucan survived the challenge with V. salmonicida., whereas 70% ofthe M-Glucan treated fish survived. The survival in the control group was 4%.

5 10 15Days after inoculafion

Figure 2. Cumulative mortality in Atlantic salmon treated with M-Glucan or saline alter challenge with V.anguillarum. Conditions and symbols as in Fig. 1. The fish were challenged by i.p. injection of 5x10"^bacteria per fish {n=40).

396 B. Koinrtscn cl al.

5 10 15 20Days after inoculafion

25

Figure 3. Cumuhuivc mortality in Atlantic salmon treated with M-Glucan or saline after challenge with Yersiniaruckeri. Conditions and symbols as in Fig. 1. The fish were challenged by i.p. injection of 10'* bacteria perfish (N-50).

In four different experiments using V. salmonicida as the pathogen, the RPP obtained withM-Glucan was higher than the RPP obtained with DL-Glucan. In these experiments, the RPPobtained with M-GIucan and DL-Glucan was 81% versus 47%, 67% versus 10%, 61% versus9%, and 53% versus 2%.

Dose response of M-Glucan

In the initial experiments, the authors injected a relatively large dose of glucan, i.e. 2nig, intoeach fish. At this glucan concentration it took about 3 weeks for the fish to develop maximalresistance. When different glucan doses were injected and the fish challenged one week laterwith V. anguillarum. the best protection was obtained with glucan concentrations of 50—200 ugper fish (Table 2). At higher and lower concentrations the protection was less.

0 5 \0 15 20Days after inoculation

Figure 4. Comparison of the protective effect of M-GIucan and DL-Glucan in Atlantic salmon. Atlantic salmonwith a mean wt of 20g were injected i.p. with a 0-2nil suspension of 2mg M-Glucan (•) or DL-Glucan (A)in saline, or with 0-2ml saline (n). The fish were challenged 3 weeks later with V. satmonicida by i.p.injection of .5x10^ bacteria per fish ( / J=50) .

Enhancement of resistance in salmon hy M-Glucan 397

Table 2. Effect of different doses of M-Glucan on the resistance of Atlantic salmon against Vibrio anguillarum.

Amount of glucaninjected per fish

01050

200600

1800

Mortality

Experiment 1

76nt"27457793

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Experiment 2

10093535374

nt"

*'' Salmon with a mean weight of about 20 g wereinjected i.p. with saline or different amounts ofM-Glucan in saline in groups of 30 individuals. Allgroups were kept in the same tank. The fish werechallenged one week later by i.p. injection of 5x10** V.anguillarum serotype OL Total mortality wasregistrated 14 days after inoculation.* nt: not tested.

Chemical composition of M-Glucan

The hexose content of M-GIucan was determined to be 97 4±5-2%. Hydrolysis of M-Glucanproduced glucose as the only monosaccharide (Fig. 5), which shows that it is a glucan. The lownitrogen content (0 45±0-01%, mean ±S.D., n=4) further confirms that the polysaccharidepreparation has a high degree of purity.

Discussion

The present work demonstrates that M-Glucan has the ability to induce protection againstthree different pathogens in Atlantic salmon. M-Glucan probably acts by activating the non-specific defence mechanisms in fish. It is important to note that the protective effect wasobtained without suspending the glucan in a mineral oil suspension, which was used in studieswith a mycobacterium as immunostimulant in fish (Olivier et al. 1985, 1986).

By the present study, fish can be added to the list of organisms which respond to fungal cellwall glucans by activation of defence mechanisms. The recognition of fungal cell wall glucans isapparently a mechanism which has developed early in the evolution of higher organisms as amechanism of defence against fungal pathogens.

Measurements of neutral hexose by the anthrone method and monosaccharides by GLC,show that M-GIucan contains only glucose. DL-Glucan has also been reported to containglucose as the sole monosaccharide constituent (DiLuzio et al. 1979). Even though M-GIucanand DL-Glucan are both insoluble polysaccharides with glucose as the only monomeric unit,the two preparations have quite different potencies in enhancing disease resistance in salmon.This shows that the structural composition of the preparations must be different, and itfurthermore demonstrates that the protective effect is not just an effect of injection of particlesinto the fish. The difference in composition between M-Glucan and DL-Glucan is likely to bedue to differences in glycosidic linkages. This aspect is currently under investigation.

DL-GIucan has been shown to be a potent stimulant of non-specific disease resistance inmice (DiLuzio et al. 1979; DiLuzio 1983; Wilhanis et al. 1983). The maximal protection using

398 B. Robertsen et al

00 COOpch

Solvent Glucose

Inositol(internalstandard)

I ) i I J L

0 5 10Retention time (min)

15

Figure 5. GLC-chromatogram of monosaccharidcs (alditol acetates) obtained by acid hydrolysis of M-GIucan.

2mg M-Glucan per fish was obtained at 3—4 weeks after injection. When salmon were injectedwith 10-1800//g M-Glucan per fish and ehalienged one week later with V. anguillarum, thebest protection was obtained with 50—200/ g glucan. At the highest glucan concentration, thefish sometimes expressed a higher mortality than in the control group. The reason may be thatwhen large amounts of glucan are injected, the phagocytic cells of the fish become overloadedwith glucan particles and are therefore less able to phagocytose bacteria for 1-2 weeks. Theprotective effect of the glucan may thus not appear before a certain digestion of glucan hasoccurred. This view is supported by the observation that the protective effect of the low glucandoses (200//g) declined from day 7 after injection (data not shown).

These results are in accordance with those of Olivier et al. (1985) who used killed myco-bacteria to induce disease resistance in rainbow trout. At doses of SO iig per fish, the myeo-

Enhancement of resistance in salmon by M-Glucan 399

bacteria indueed resistance, whereas 1 mg per fish resulted in enhanced susceptibility. Injectionof particles like latex or silica also increased the susceptibility of rainbow trout to infection.This effect was interpreted as impairment of the macrophage function by high doses ofparticles. The increased disease resistance in rainbow trout obtained with mycobacteria isapparently associated with activation of the maerophages, because maerophages from rainbowtrout injected i.p. with Freund's complete adjuvant (killed mycobacteria in light mineral oil)killed virulent Aeromonas salmonicida more efficiently than maerophages from trout whichhad been injected with glycogen (Olivier et al. 1986). Recently, Graham, Jeffries & Secombes(1988) showed that maerophages from rainbow trout injected with killed cells of an avirulentstrain oi A. salmonicida suspended in Freund's incomplete adjuvant had a higher ability to killa virulent strain of A. salmonicida than maerophages from control fish which had been givensaline. However, in this work, any correlation between the effect on maerophages and thedegree of disease resistance in live fish was not investigated. Maerophages also play a key rolein the protection produced by yeast glucan in mice (DiLuzio 1983; Williams et al. 1983), butthe precise mechanism of action is not known. However, it has been shown that yeast glucanactivates mouse maerophages in vitro by increasing their size, spreading abiUty, ability toincorporate ^"^C-D-glucosamine and ability to lyse tumour cells (Seljelid, Bogwald & Lundwald1981; Bogwald, Johnson & Seljelid 1982). It has also been shown that yeast glucan induces theproduction of interleukin 1 and interleukin 2 in mice (Sherwood, Williams, McNamee, Jones,Browder & DiLuzio 1987) and that it enhances the number of neutrophils in the blood(Williams, Sherwood, Browder, McNamee, Jones, Rakinic & DiLuzio 1988). The presentauthors are currently investigating what kind of defence mechanisms M-Glucan elicits inAtlantic salmon.

Immunostimulatory compounds may be used to increase the general resistance in farmedfish or as adjuvants in combination with fish vaccines. The application of highest potential is asadditives in the feed. Therefore, the main effort in this research is directed towards the use ofM-Glucan or derivatives thereof in fish feed.

Acknowledgments

The authors wish to thank Anne Ramstad and Sigrun Kjelbotn at the veterinary station forcontract research in fish diseases, Vikan, for technical assistance with the challenge experi-ments. TTiis work was supported by grants from Mackzymal A/S, Troms0, Norway, andPhillips Petroleum Company, Norway.

References

Albersheim P., Nevins D. J., English P. D. & Karr A. (1967) A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydrate Research 5, 340-345.

Bogwald J., Johnson E. & Seljelid R. (1982) The cytotoxic effect of mouse maerophages stimulated in vitro by a/3-1,3-D-giuean from yeast cell walls. Scandinavian Journal of Immunology 15, 297-304.

Darvill A. G. & Albersheim P. (1984) Phytoalcxins and their elicitors — a defence against microbial infectionsin plants. Annual Review of Plant Physiology 35, 243—275.

Davis J. F. & Hayasaka S. S. (1984) The enhancement of resistance of Ihe American eel. Anguitla rostrata LeSueur, to a pathogenic bacterium, Aeromonas hydrophila, by an extract of the tunicate, Ecteinascidiaturbinata. Journal of Fish Diseases 7, 311—316.

DiLuzio N. R., Williams D. L., McNamee R. B., Edwards B. F. & Kitahama A. (1979) Comparative tumor-inhibitory and anti-bacterial activity of soluble and paniculate glucan. hiternaiional Journal of Cancer 24,773-779.

400 /^ Robcrtscn ct :il.

DiLuzio N, K. ( U)S,\) Immunophnrmaeology of glucan: a broad spectrum enhancer of host defence meehanisms.Trends in rharnuicoloiiical Sciences 4, 344 —.M7.

DiLu;cio N. R. (1985) Update on the immunomoduiatiiig aetivilies of glucans. Springer Seminars in (mmuno-(Httholoi^y 8, -\S7-4(H).

Dische Z. (I )()2) Color reaction of carbohydrates, In: Methods in Carbohydrate Chemistry, Vol. 1 (cd. by R. L.Whisller .v;: M. L. Wollrom), pp. 478-481. Aaulemic Press, New York.

Duftus J. H., Lcvi C. &. Manners D. J. (1982) Yeast cell-wall glueans. Advances in Microbial Physiology 23,151-181.

Feniehel R. L. & Chirigos M. A. (1984) Immune Modulation Agents and Their Mechanisms. Mareel Dekker.Ine.. New York.

Fletcher T. C. (1982) Non-spceitic defence mechanisms of tish. Developmental and Comparative Immunology 2,123-132 (Suppl).

Graham S., Jeffries A. H. & Secombes C. J. (1988) A novel assay to dctcet maerophage bactericidal activity intish: factors influencing the killing of Aeromonas salmonicida. Journal of Fish Diseases 11, 389-396.

Holm K. O. & J0rgensen T. (1987) A successful vaccination of Atlantic saimon, Salmo salar L., against 'Hitradisease' or coldwater vibriosis. Journal of Fish Diseases 10, 85-90.

Kitao T. & Yoshida Y. (1986) Effect of an immunopolentiator on Aeromonas salmonicida infection in rainbowtrout {Salmo gairdneri). Veterinary Immunology and Immunopathology 12, 287—296.

Lamers C. H. J. (1985) The Reaction ofthe Immune System of Fish to Vaccination. PhD thesis. AgriculturalUniversity. Wagcningen, Netherlands.

Olivier G., Eaton C. A. & Campbell N. (1986) Interaction between Aeromonas salmonicida and peritonealrnacrophages of brook trout {Salvelinus fontinalis). Veterinary Immunology and Immunopathology 12,223-234.^

Olivier G., Evelyn T. P. T. & LaUier R. (1985) Immunity to Aeromonas salmonicida in eoho salmon{Oncorhynchus kisutch) induced by modified Freund's complete adjuvant: its non-specific nature and theprobable role of macrophages in the phenomenon. Developmental and Comparative Immunology 9.419-432.

Rijkers G. T., van Oosterom R. & van Muiswinkel W. B. (1981) The immune system of cyprinid fish.O.xytetracyclinc and the regulation of humoral immunity in carp {Cyprinus carpio). Veterinary Immunologyand Immunopathology 2, 281—290.

Rosenberger R. F. (1976) The cell wall. In: The Filamentous Fungi, Vol. 2, Biosynthesis and Metabolism (ed. byJ. E. Smith & D. R. Berry), pp. 328-334. Edward Arnold, London.

Sherwood E. R., Wilhams D. L.. McNamee R. B., Jones E. L., Browder L W. & DiLuzio N. R. (1987)Enhancement of interleukin-1 and interlcukin-2 production by soluble glucan. Internationa! Journal ofImmunopharmacology 9, 261 — 267.

Scljelid R., B6gv»'ald J. & Lundwald, A. (1981) Glyean stimulation of macrophages in vitro. Experimental CellResearch 131, 121-129.

Unestam T. & Soderhall K. (1977) Soluble fragments from fungal cell walls elicit defence reactions in crayfish.Nature 267, 45-46.

Williams D. L., Browder I. W. & DiLuzio N. R. (1983) Immunothcrapeutic modification of Escherichia coli-indueed experimental peritonitis and bacteremia by glucan. Surgery 93, 448-454.

Williams D. L., Sherwood E. R., Browder L W., MeNamec R. B., Jones E. L., Rakinic J. & DiLuzio N. R.(1988) Effect of glucan on neutrophil dynamies and immune funetion in Escherichia coli peritonitis. Journalof Surgical Research 44, 54—61.

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