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Effects of supplementing the feed to Atlantic cod Gadus morhua / fry with lactic acid bacteria and immuno-stimulating peptides during a challenge trial with Vibrio anguillarum
Asbjrn Gildberg ) , Helene Mikkelsen
Norwegian Institute of Fisheries and Aquaculture, 9005, Troms, Norway Accepted 8 June 1998

Abstract Atlantic cod Gadus morhua. fry were reared on a commercial feed supplemented with Carnobacterium diergens, isolated from the intestines of Atlantic cod and Atlantic salmon Salmo salar . alone or in combination with immuno-stimulating peptides. In vitro experiments showed that culture filtrates from the isolates from cod and salmon inhibited the growth of Vibrio anguillarum. After 3 weeks of feeding, the fish were challenged by bath exposure to V. anguillarum 10 7rml, 1 h.. Twelve days after infection significant p - 0.05. reduced cumulative mortality was recorded in fish given feed supplemented with lactic acid bacteria isolated from Atlantic salmon and with immuno-stimulating peptides. No synergistic or cumulative effects were achieved by combining lactic acid bacteria and immuno-stimulating peptides. Four weeks after infection the same cumulative mortality 8084%. was reached in all groups. Both strains of lactic acid bacteria could colonize the internal mucus layer of the cod fry pyloric caeca, and a significant number of the bacteria survived the passage of the whole gastrointestinal tract. q 1998 Elsevier Science B.V. All rights reserved.
Keywords: Immuno-stimulating peptides; Probiotics; Carnobacterium diergens; Atlantic cod; Intestinal colonisation

1. Introduction In recent years efficient vaccines have been developed against many of the major bacterial diseases in aquaculture. Nevertheless, considerable efforts are still being made
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Correspondence author. Tel.: q47-776-29-000; Fax: q47-776-29-100; E-mail: asbjg@fiskforsk.norut.no

0044-8486r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 9 8 . 0 0 2 9 6 - 8

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to develop alternative or supplementary methods to improve fish health. Among such methods, the prophylactic use of immuno-stimulants and probiotics has attracted particular interest Raa, 1996; Ring and Gatesoupe, 1998.. One obvious reason for this is that such approaches can be implemented at larval and early fry stages where vaccines can not be used. At these stages mortality can be high due to proliferation of opportunistic pathogens even where the initial infection pressure is low Muroga et al., 1987.. In such situations minor improvements of the non-specific immune response system or the intestinal microbial flora may reduce the mortality significantly. It has now been established that several species of lactic acid bacteria LAB. are part of the natural intestinal flora of healthy fish Ring and Gatesoupe, 1998., and it is well known that LAB often produce bacteriocins which may inhibit the growth of Gramnegative fish pathogens. Apparently, LAB are rarely present in juvenile fish reared on artificial feed, but may become dominant in the intestinal flora if they are supplemented in the feed Gildberg et al., 1995, 1997.. It has been shown that species of Carnobacterium isolated from fish can produce bacteriocins which inhibit the growth of various pathogens Pilet et al., 1995; Joborn et al., 1997.. In a previous challenge experiment no protection was obtained against infection by Aeromonas salmonicida after giving Atlantic salmon fry a feed supplemented with Carnobacterium diergens isolated from mature salmon Gildberg et al., 1995.. In a similar experiment some protection against classical vibriosis was achieved in Atlantic cod fry when they were given a feed containing C. diergens isolated from the intestines of cod Gildberg et al., 1997.. Recently it was shown that low molecular weight peptides may stimulate the activity of fish macrophages Bgwald et al., 1996; Gildberg et al., 1996.. Both in vivo and in vitro experiments showed that low molecular weight peptide fractions from fish protein hydrolysates stimulated oxidative burst- and morphological cell reactions in head kidney leukocytes from Atlantic salmon. Reports of challenge experiments with administration of immuno-stimulating peptides to fish are rare, but protection of rainbow trout Oncorhynchus mykiss . against furunculosis was reported to have been achieved after intraperitoneal injection of low molecular weight peptides Kitao and Yoshida, 1986.. In the present study potential probiotic feeds were made by supplementing commercial dry feed with two different strains of C. diergens; one isolated from Atlantic codand the other from Atlantic salmon intestines. In addition, feed supplemented with immuno-stimulating peptides and feeds where peptides and LAB were supplemented together were made. Atlantic cod fry reared on these different diets were challenged by infection with Vibrio anguillarum. The major aims with this experiment were to compare the effects of the two different strains of C. diergens regarding intestinal colonisation and probiotic protection and to detect any eventual synergistic effects between immuno-stimulating peptides and LAB. 2. Materials and methods 2.1. Experimental design Atlantic cod Gadus morhua. fry about 2000. were obtained from Parisvatnet The Marine Res. Inst., Bergen.. About 1200 apparently healthy and even sized fish were

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selected and stocked in six square tanks about 200 fish in each tank. containing 250 l aerated sea water at 108C. The water exchange rate was about 4 lrmin. The fish were held for 2 weeks and fed ad libitum on a commercial dry pellet feed FK Marine pellet, f 2.0, batch no. 961210., later referred to as the control feed. After 2 weeks samples of at least 30 fish from each tank were weighed and returned to their tanks. Feeding with various modifications of the control feed was initiated in five of the six tanks; this was continued throughout the rest of the experiment. After 3 weeks of rearing on the different diets, further samples of fish ) 30 from each tank. were weighed to determine the specific growth rate SGR.. At this stage 10 fish from each tank were removed for analysis of the intestinal microbial flora. Two days after this weighing, the remaining fish about 190 in each tank. were bath challenged by a strain of V. anguillarum LFI 1243. isolated from infected Atlantic cod. About 10 12 colony forming units CFU. were added to each tank after the water volume of the tanks had been reduced to 100 l, yielding an initial concentration of viable bacteria of about 10 7rml. During the challenge period the water exchange was stopped, and oxygen was supplied above 80% saturation. to each tank. After 1 h the water volumes were increased to 250 l, and normal water exchange was re-established. The challenge experiment proceeded for 4 weeks, dead fish being removed once a day. Samples from the kidney of representative dead fish were plated on blood-agar plates containing 2% NaCl for isolation of bacteria. Classical vibriosis was diagnosed on the basis of the re-isolation of typical haemolytic colonies in pure culture. The diagnosis was confirmed by testing colonies with a rapid agglutination test using Mono-Va from BioNor, Skien, Norway. At the end of the experiment, intestines from 10 surviving fish from each tank were subjected to microbial analysis. 2.2. Statistical analysis To evaluate the significance of the differences in mortality between fish given test diets and control feed, an approximation to the standard normal distribution was used Bhattacharyya and Johnson, 1977.. This test has been found to be convenient for statistical analyses of challenge experiments with a large number of fish in each group Bgwald et al., 1992; Gildberg et al., 1995.. When values of p - 0.05 were achieved, the differences were regarded to be significant. 2.3. Preparation of feed The control feed served as the basis for all the five experimental diets. The experimental diets were prepared by absorbing suspensions of LAB isolated from fish intestines andror a solution of immuno- stimulating peptides from a fish protein hydrolysate Gildberg et al., 1996.. The absorption was achieved by pumping the liquid through a syringe tip f 0.6 mm. onto the dry feed pellets which were placed in a tilted rotating round bottle 2 l. with a shovel inside. The best results suitable consistency, and high survival of bacteria. were obtained when portions of 200 g of dry feed were sprayed with 35 ml of liquid. After spraying, the feed was airdried in a vent hood at room temperature overnight, and the moisture content was reduced to about 5%, which

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was slightly less than the initial moisture content of the commercial feed before spraying. Finally, 200 g portions of the feed were vacuum packed and stored at y308C until used. The test diets contained either viable LAB alone, immuno-stimulating peptides alone or mixtures of LAB and immuno-stimulating peptides. The potential probiotic LAB used were two strains of C. diergens isolated from the intestines of Atlantic cod and Atlantic salmon. The bacteria added to the feed had been washed and re-suspended in saline water and yielded a viable count of about 10 8rg of dried and vacuum packed feed. The peptide fraction used was composed of acid peptides obtained by ion exchange chromatography of the - 3000 d fraction of a cod stomach hydrolysate Gildberg et al., 1996.. The peptone solution 20 mgrml. was adjusted to pH 2.0 by hydrochloric acid and applied on a BioProcess column Pharmacia Biotech. containing S-Sepharose fast-flow gel Pharmacia Biotech. equilibrated at pH 2.0 by 20 mM HCl. The column was eluted by 20 mM citraterHCl buffer pH 2.5. and the fraction eluting in the pH range 2.12.4 was collected. It was then concentrated by freeze-drying and applied as the peptide supplement in the experimental diets. The amount of peptide added corresponded to about 0.01% of the feed weight. The test experimental diets were given the following identifications: Feed supplemented with lactic acid bacteria from cod, LABC; lactic acid bacteria from salmon, LABS; the peptide fraction alone, Peptide; and finally the two feeds supplemented with both LAB and peptide; LABCq P and LABSq P, respectively. 2.4. Preparation of the LAB cultures Two strains of C. diergens isolated from the intestines of medium size about 1 kg. Atlantic cod and Atlantic salmon were chosen as potential probiotics. The bacteria were isolated from the inside of thoroughly washed segments of the upper intestines Strm, 1988., and classified according to species level using the criteria given by Hamnes et al., 1992. The salmon bacterium, which initially was erroneously identified as a strain of Lactobacillus plantarum, has proven to inhibit the growth of V. anguillarum in in vitro culture experiments Strm, 1988.. The bacteria were grown 3 days at 128C in shaken bottles with FMg, full medium Be, 1980. composed of Bacto trypton 0.5%., K 2 HPO4 0.2%., glucose 0.5%. and 1% of each of a standard mineral-, vitamin- and nucleotidesolution Ford et al., 1958.. The cultures were centrifuged 15 min, 4000 = g ., and the pellets resuspended in sterile saline water to OD600nm s 2.02.5, before being sprayed on the pellets. 2.5. Microbial analysis The analysis of LAB in the intestinal tract and faeces was done on two parallel samples of five fish from each tank. The samples of the intestines were divided in two parts, one part included only the pyloric caeca pooled from the five fish, whereas the second part consisted of excised washed intestines excluding the pyloric caeca. Also the faeces from the same five fish were pooled and analyzed. The gut contents were washed out into two baths of sterile 50% artificial seawater Baumann and Baumann, 1981.. The

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intestine- and the pyloric caeca fractions were homogenized in Stomacher bags Stomacher, Lab-Blender 400. with 10 ml sterile 50% artificial seawater. The presumptive number of LAB was determined using Plate Count Agar without NaCl PCA, Oxoid.. The plates were incubated at 128C for 2 weeks. C. diergens were the only bacteria present that could grow on plate count agar without salt, where they appeared as white-greyish colonies. To verify this, some of the C. diergens colonies were tested in an enzyme linked immunosorbent assay ELISA. Espelid et al., 1988.. Bacteria were fixed in 0.5% formalin and washed in PBS before immobilisation on 96-wells microtiterplates Maxisorp, Nunc, Denmark. pretreated with poly-L-lysine 5 mgrml, MW 200 000 d, Sigma, USA.. Nonspecific binding was blocked with 1% BSA, before polyclonal rabbit antibodies against each of the two strains were added to the wells and incubated for 1 h. After washing, alkaline phosphate conjugated anti-rabbit-Ig Dakopatts, Denmark. was allowed to bind and finally p-nitrophenylphosphate 1 mgrml Sigma. was added as a substrate. Antibody binding was measured spectrophotometrically at 405 nm. 2.6. Immunohistochemistry C. diergens was detected by a horseradish-peroxidase-AEC 3-amino-9-ethylcarbazole.-complex. Three fish from each experimental group were killed with a blow to the head, and the abdomens were immediately cut open with a sterile scalpel. Samples of the intestines were divided in three parts; pyloric caeca, upper- and lower intestines. The samples were fixed for at least 12 h in 4% buffered formalin with 1% cetylpyridine and dehydrated through a graded ethanol series Citadel 1000, Shanon., and embedded in paraffin. Sections, 4 mm thick, were cut and incubated at 588C for 30 min before they were dewaxed in xylene, rehydrated through a graded ethanol series, and brought to Tris-buffered saline, TBS 50 mM TrisrHCl, pH 7.8 with 0.15 M NaCl.. Endogen peroxidase activity was blocked by incubation with H 2 O 2 for 10 min at room temperature. The rabbit antisera against C. diergens were diluted 1:500 in TBS with 0.5% skimmed milk, and incubated for 1 h at room temperature. Visualisation of C. diergens was obtained with Ultra Tek HPR anti-rabbit kit Scytek Laboratories, Logan, USA. according to the manufacturers instructions. Finally, the sections were counterstained with Mayers haematoxylin, mounted with glycerol 50% in TBS., and examined by light microscopy Nikon Optiphot-2.. 2.7. Bacterial growth inhibition studies The presence of growth inhibitory substances in C. diergens culture against V. anguillarum LFI 1243 were tested using sterile culture supernatant Joborn et al., 1997.. A total of 10 ml cultures of the two strains of C. diergens were grown for 3 days at 128C in FMg with 1.5% NaCl and pH 6.5 Fmgs.. After centrifugation 10 min, 13 000 = g ., the culture supernatants were filtersterilized 0.2 mm. and serially diluted in two-fold steps with FMgs broth in sterile microtiter plates Costar U bottom: 50 ml per well.. Then 50 ml FMgs broth were added to each well. Growth of the fish pathogen V. anguillarum in different dilutions of culture supernatant was measured by inoculating

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samples of the pathogen culture in its logarithmic growth phase. Culture samples 3 ml., diluted with FMgs to an optical density of 0.3 at 600 nm, were inoculated in each well containing 100 ml medium with two-fold dilutions of the culture filtrates. Pure FMgs inoculated with the pathogen were used as a control, and the plate was incubated at 248C for 24 h in a microtiter spectrophotometer Spectramax plus, Molecular Devices, USA..

3. Results and discussion The microtiter plate experiment showed that the growth of V. anguillarum was inhibited completely by undiluted culture filtrates of both strains of C. diergens, whereas about 60% inhibition was obtained with the first two-fold dilution of both culture filtrates. Although the inhibitory effect was not high, it indicated that both strains of C. diergens produced chemical substances which may reduce the infection pressure of V. anguillarum if they colonize the intestinal tract of the cod fry. This is in accordance with results obtained by Joborn et al. 1997. showing that a Carnobacterium sp. isolated from salmon inhibited the growth of both V. anguillarum and A. salmonicida in intestinal fish mucus. The pre-challenge feeding trial Table 1. shows that the specific growth rates of fish given different diets were fairly uniform and apparently normal for cod fry of this size Gildberg et al., 1997.. Fig. 1 shows that the number of viable LAB was relatively high 10 4 CFUrg. in the pyloric caeca of fish given feed supplemented with C. diergens. No LAB were detected in fish given diets where LAB were not added. Apparently the colonisation of LAB in the intestines was lower and less consistent than in the pyloric caeca. LAB were not found in the intestines of fish given feed supplemented with lactic acid bacteria from cod LABC. alone, although average levels of LAB were detected in the pyloric caeca of the same fish. It is interesting to observe that C. diergens isolated from salmon apparently has the same ability to colonize the intestinal tract of cod fry as the strain isolated from cod intestines. Thus, in this case there is apparently no host specificity giving preference for colonisation by the strain isolated from cod. Fig. 2 clearly verifies colonisation of C. diergens in the internal mucus layer of the pyloric caeca. A similar distinct colonisation could not be observed in the mucus layer of the intestines. These

Table 1 Growth of cod fry during the initial 3 weeks of feeding before infection Feed Control LABC LABS Peptide LABCqP LABSqP Average initial weight g. 6.1 5.7 5.8 5.8 6.2 5.6 Average weight after 21 days g. 10.0 10.0 10.2 10.8 10.0 9.0 SGR %rday. 2.35 2.68 2.69 2.96 2.28 2.26

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Fig. 1. Number of viable lactic acid bacteria in pyloric caeca and intestines of Atlantic cod Gadus morhua. fry after 3 weeks of rearing on feed containing Carnobacterium diergens isolated from the intestines of mature Atlantic cod LABC. and Atlantic salmon LABS.. Feeds also containing added immuno-stimulating peptides are indicated by qP.

observations strengthen the earlier suggestion that in cod fry the pyloric caeca seems to be more accessible to colonisation by LAB than the intestines Gildberg et al., 1997.. In all the fish given diets supplemented with LAB, the faeces contained about 10 6 CFU of LAB per gram, whereas no LAB were detected in fish given diets where LAB had not been added. Although the content of viable LAB in the faeces was only 1% of the content of the feed, these results clearly demonstrated that a significant amount of both strains of C. diergens supplied orally survived the passage through the gastrointestinal tract of Atlantic cod fry. The challenge experiment Fig. 3. showed that none of the experimental diets provided reduced mortality. Four weeks after infection the cumulative mortality had stabilized at about 80% in all the tanks. In the initial phase, however, significant differences p - 0.05. were observed Fig. 4.. Twelve days after infection, fish given feed supplemented with lactic acid bacteria from salmon LABS. and fish given feed supplemented with peptides had significantly lower cumulative mortality than fish given control feed. No protection was obtained for fish given feed supplemented with lactic acid bacteria from cod LABC.. This does not correspond with earlier results which showed some probiotic effect of this strain in a similar challenge experiment with cod fry Gildberg et al., 1997.. The explanation for this may be that the bacteriocin production can be inducible and may not occur if the bacteria are not frequently challenged with competitors Schrder et al., 1980.. The peptide fraction added to the feed corresponded to peptide fractions which have induced considerable in vitro activation of salmon leukocytes Gildberg et al., 1996..

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Fig. 2. Immunostaining pictures of Atlantic cod G. morhua. fry pyloric caeca. a. Fish given control feed. b. Fish given feed containing C. diergens isolated from the intestines of mature Atlantic cod. C. diergens is visualized using rabbit antisera against the bacterium 1:500 dilution., and anti-rabbit horseradish-peroxide conjugated immunoglobulins.

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Fig. 3. Cumulative mortality of Atlantic cod G. morhua. fry, given different experimental diets, as a function of time after infection by Vibrio anguillarum. Control feed v ., LABC I., LABS B., Peptide '., LABCqP `. and LABSqP ^..

The present challenge experiment indicated that some stimulation of the non-specific immune defence system occurred in the cod fry after oral administration of the peptides. This corresponds with the results from initial experiments not published., which also

Fig. 4. Comparison of cumulative mortality of Atlantic cod G. morhua. fry 12 and 24 days after infection with V. anguillarum. Columns marked with asterisks). represent cumulative mortality significantly p- 0.05. different from the mortality of fish given control feed.

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indicated that the amount of peptide in the feed was very critical. If too high a level of such peptides or other immuno-stimulants are added to the feed they may affect the non-specific immune system negatively Raa, 1996; Gildberg et al., 1997.. Synergistic or cumulative effects of LAB and peptide immune stimulants were not obtained in this experiment. The marginal probiotic and immuno-stimulatory effects achieved in this challenge experiment indicate that oral administration of probiotics or immuno-stimulatory peptides do not give efficient protection of fish fry when they are exposed to high levels of highly virulent pathogens. This does not exclude the possibility though, that such methods may be of considerable importance under normal rearing conditions in the presence of moderate levels of opportunistic bacteria. Consequently, a more convenient method for detection of probiotic effects would be to compare the mortality of fish given different feeds in large scale feeding trials, where the fish is not exposed to high concentrations of aggressive pathogens.

Acknowledgements The authors wish to thank Jarl Bgwald for constructive criticism of the manuscript and Elin Sandaker for clever technical assistance. Financial support from The French Norwegian Foundation for Scientific and Technical Research FNS. and The Norwegian Research Council is acknowledged.

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