Aquaculture 302 (2010) 1–18

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Aquaculture

j ourna l homepage: www.e lsev ie r.com/ locate /aqua-on l ine

Review

The current status and future focus of probiotic and prebiotic applicationsfor salmonids

Daniel L. Merrifield a,⁎, Arkadios Dimitroglou a, Andrew Foey b, Simon J. Davies a, Remi T.M. Baker a,Jarl Bøgwald c, Mathieu Castex d, Einar Ringø c

a Aquaculture and Fish Nutrition Research Group, School of Marine Science and Engineering, Marine Institute, University of Plymouth, UKb School of Biomedical and Biological Sciences, University of Plymouth, Plymouth, UKc Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, University of Tromsø, Norwayd Lallemand Animal Nutrition, Blagnac, France

⁎ Corresponding author. Tel.: +44 1752 584 877; faxE-mail address: daniel.merrifield@plymouth.ac.uk (D

0044-8486/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.aquaculture.2010.02.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 November 2009Received in revised form 4 February 2010Accepted 5 February 2010

Keywords:DiseaseSalmonTroutSynbioticMicrobiotaHealth

Salmonids are an important contributor to fish production in many countries. Concerted research effortshave concentrated on optimising production with eco-friendly alternatives to the therapeutic use ofantimicrobials. Probiotics and prebiotics offer potential alternatives by providing benefits to the hostprimarily via the direct or indirect modulation of the gut microbiota. Suggested modes of action resultingfrom increased favourable bacteria (e.g. lactic acid bacteria and certain Bacillus spp.) in the gastrointestinal(GI) tract include the production of inhibitory compounds, competition with potential pathogens, inhibitionof virulence gene expression, enhancing the immune response, improved gastric morphology and aidingdigestive function. The application of probiotics and prebiotics may therefore result in elevated health status,improved disease resistance, growth performance, body composition, reduced malformations and improvedgut morphology and microbial balance.Current research demonstrates successful proof of these concepts and a foundation for applications insalmonid aquaculture. However, application strategies applied in current studies are varied and oftenimpractical at industrial level farming; thus, it is difficult to plan an effective feeding strategy for commerciallevel applications. Future studies should focus on providing practical industrial scale applications.Additionally, from a scientific perspective we must have a better understanding of the mucosal–bacterialinteractions which mediate the host benefits in order to achieve optimal utilisation.

: +44 1752 584950..L. Merrifield).

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© 2010 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Endogenous microbiota, mucosal tolerance and development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23. Probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.1. Probiotic selection criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33.2. Mode of action and reported probiotic benefits in salmonids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33.3. Immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.4. Disease resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.4.1. Viral diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.4.2. Bacterial diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.4.3. Disease challenge methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.5. Effect of probiotics on gut microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.6. Gastrointestinal morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.7. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.8. Feeding strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3.8.1. Choosing a probiont. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.8.2. Supplementation form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2 D.L. Merrifield et al. / Aquaculture 302 (2010) 1–18

3.8.3. Vector of administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.8.4. Dosage level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.8.5. Supplementation duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.9. Issues with industrial level scale-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.10. Future probiotic work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

4. Prebiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114.1. Mannanoligisaccharides (MOS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.2. Inulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134.3. Other prebiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134.4. Future prebiotic work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

5. Synbiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146. Concluding remarks and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1. Introduction

Although there is now global recognition that aquacultureproduction is expanding to a wide diversity of cultured finfish,salmonids remain an important contributor to fish production inmany countries. Atlantic salmon (Salmo salar L.) and rainbow trout(Oncorhynchus mykiss W.) are reared principally in Norway, Scotlandand Chile with some output arising from Canada, the USA, regions ofEurope and to a lesser extent Australia and New Zealand. Total globalproduction of salmonids was reported to exceed 2.2 million mt in2007 (FAO, 2009). These species attract high market prices and are astable source of high quality fish due to consumer acceptance forquality seafood. Salmon in particular are noteworthy for theircharacteristic pink-reddish flesh pigmentation (Davies, 2008) andversatility with respect to processing into a wide range of foodproducts. Rainbow trout is also a popular salmonid fish species andhas received great attention in terms of importance to aquacultureand remain a core of inland fish production in many countriesthroughout the world. It should also be recognised that native speciesof brown trout (Salmo trutta) are important especially in terms oftheir contribution to recreational fisheries and angling. Additionally,developments in rearing techniques for Arctic charr (Salvelinusalpinus) have increased interest in commercial farming and conse-quently global production has risen to over 2000 mt in 2007 (Joblinget al., 1993; FAO, 2009). Collectively, there has been an abundance ofscientific literature underpinning the genetics, nutrition, health anddisease issues concerning the development of the salmonid aquacul-ture industry.

Given the importance of nutrition inmaintaining the health of fish,with respect to nutritional involvement on immuno-competence anddisease resistance, as well as its role in stress mediation, there is agrowing trend towards exploring dietary components of a non-nutritional nature to provide various functional attributes. This hasbeen compounded by the constraints of employing antibiotics in theaquaculture industry, as reflected by the EU moratorium on thebanning of antibiotic growth promoters in animal feeds, including fish(Regulation, EC No, 1831).

There have been numerous investigations on salmon and trout toevaluate the feasibility of supplementing diets with a range ofpotentially probiotic bacteria. Several general reviews have beenpublished over the past decade summarising the latest availableliterature (Ringø and Gatesoupe, 1998; Gatesoupe, 1999; Ringø andBirkbeck, 1999; Vershuere et al., 2000; Irianto and Austin, 2002a; Ringø,2004; Burr et al., 2005; Balcázar et al., 2006a;Gram and Ringø, 2005;Ringø et al., 2005; Gatesoupe, 2007; Kesarcodi-Watson et al., 2008;Wang et al., 2008a). Furthermore, specific reviews have focused onlarvae (Gomez-Gil et al., 2000; Vine et al., 2006; Tinh et al., 2008),

shrimp (Farzanfar, 2006; Ninawe and Selvin, 2009), shellfish (Balcázaret al., 2006b) along with reviews specific to applications for Indian(Panigrahi and Azad, 2007) and Chinese aquaculture (Qi et al., 2009).However, to the authors' knowledge, no reviews have been put forwardto summarise the effects of probiotics on salmonids. Additionally, withthe growing interest and assessment of prebiotic applications for fish, itis pertinent to review the presentfindingswith regards to salmonidfish.

The aim of this review is to evaluate the literature currentlyavailable regarding the use of probiotics and prebiotics (collectivelyreferred to as “biotics” hereafter) on salmonids. Specific emphasis isplaced on highlighting application strategies on a practical basis andpotential future research. In order to discuss these issues wemust firstexamine the complex microbe–host interactions within the gut,which ultimately influence the health and development of the host.

2. Endogenous microbiota, mucosal tolerance and development

The immune system of teleost fish appears to be an efficient meansby which the host protects itself upon pathogenic challenge. But notall microbes represent a pathogenic threat; resident commensalmicrobes help maintain efficient functioning of the gut by supportinggut mucosal barrier function: mounting efficient immune responsesto pathogens that break through barrier defences or maintainingtolerance (i.e. immune non-responsiveness) to luminal contentswhich allow for nutrient absorption. The inter-relationship betweengut mucosal epithelial cells, mucus, anti-microbial products, com-mensal organisms resident in the gut and immune cells in themucosa/sub-mucosa are vital for the health and well-being of the fish.These interactions prime regulatory mechanisms which result inmucosal tolerance. From our understanding of mammalian systems,this induction of tolerance may result from selective antigen tasting ofluminal contents by specialised antigen presenting cells (eitherepithelial cells or dendritic cells) which prime regulatory T cells,which, in turn, suppress effector T cell responses. These suppressiveresponses are broken upon sensing of danger signals, presented uponpathogen invasion of mucosa/sub-mucosa (Mason et al., 2008). Nosuch regulatory T cells have been characterised as yet, in teleost fish.The characterisation of the regulatory cytokines (anti-inflammatory)IL-10 and TGFβ, however, is suggestive that such mechanisms may, infact, exist. Endogenous commensal microbiota play an important rolein tolerance induction versus immune activation decisions. Although agreat deal of further research into these complex interactions isrequired, Gómez and Balcázar (2008) recently provided a review ofour present level of understanding on the interactions between gutmicrobiota and innate immune system of fish.

Our knowledge of the importance of the endogenous microbiota onmucosal development and maturation of the immune system is

3D.L. Merrifield et al. / Aquaculture 302 (2010) 1–18

expanding, in part, due to recent studies using zebrafish (Danio rerio).The zebrafish has become an integral model species responsible formuch of our understanding of the genetics underpinning fishdevelopment and functionality (DahmandGeisler, 2006). Furthermore,several studies have demonstrated the importance of the complexmicrobe–host interactions on gastric development (Rawls et al., 2004,2007; Bates et al., 2006). For example, zebrafish gene expression hasbeen demonstrated to be influenced bymicrobial colonisation, assessedby comparing conventionally reared and germ-free zebrafish larvae(Rawls et al., 2004). Rawls et al. (2004) observed that microbiotastimulated intestinal epithelial proliferation and influenced enterocytemorphology. Furthermore, expression of serum amyloid A1 andcomplement component 3 genes were elevated in conventionallyreared larvae compared to germ-free larvae. As certain genes werealways expressed, irrelevant of the bacterial coloniser, and theexpression of other genes was bacteria-specific, the authors concludedthat a subset of zebrafish genes were responsive to factors present onlyin a subset of bacterial groups. Additionally, Bates et al. (2006) reportedthat the gut epithelial mucosa failed to differentiate fully in germ-freezebrafish larvae. A lack of brush border alkaline phosphatase activity,immature patterns of glycan expression and a distinct reduction ofgoblet and enteroendocrine cells was observed in germ-free larvaecompared to conventionally reared larvae. However, reintroduction ofmicrobiota reversed these phenotypic changes.

Therefore, manipulation of endogenous microbial populations byprobiotic or prebiotic supplementations could have appreciable effectson immunoregulatory mechanisms behind gut mucosal tolerance.

3. Probiotics

The word probiotic is constructed from the Latin word pro (for) andthe Greekword bios (life) (Zivkovic, 1999). The definition of a probioticdiffers greatly depending on the source, but the first generally accepteddefinition was proposed by Fuller (1989) as “…a live microbial feedsupplement which beneficially affects the host animal by improving itsmicrobial balance”. Given the nature of fish farming and the fact thatwater harboursmicrobial communities it is accepted that wemust havea distinctive definition for aquatic animals as opposed to that proposedby Fuller (1989) for terrestrial animals. The evolution of the definitionfor aquaculture throughout the 1990s is discussed by Gomez-Gil et al.(2000). During this period the definition was refined and newterminology for microbial applications in aquaculture were proposed.Microbes that are antagonistic to pathogens, but are not found to bepresent, either transiently or residentially, in the gastrointestinal (GI)tract, have been termed as biocontrol agents (Maeda et al., 1997;Moriarty, 1998). Microbial applications that improve water quality bythe breakdown of waste or pollutants have been termed bioaugmenta-tion or bioremediation (Moriarty, 1997, 1998; Gatesoupe, 1999).Thereafter, many authors proposed definitions for probiotics inaquaculture that were inclusive of administration via rearing waterbut tended to restrict applications tomicrobes thatwere associatedwithhealth promotingproperties (Spanggaardet al., 2001; Irianto andAustin2002a). Contrary to these definitions, somedefinitions donot focus onlyon health benefits (Moriarty, 1998; Gram et al., 1999; Vershuere et al.,2000; Farzanfar, 2006). Whilst many people now refer to all of thesemicrobial applications as probiotic treatments (sensu lato), it isimportant to distinguish the differences between them. If we were tomerge all of the proposed definitions it would appear that a probioticapplication for aquaculture is a live, dead or component of a microbialcell that when administered via the feed or to the rearingwater benefitsthe host by improving either disease resistance, health status, growthperformance, feedutilisation, stress responseor general vigour,which isachieved at least in part via improving the hostsmicrobial balance or themicrobial balance of the ambient environment.

If we are to restrict the benefits to the host to improvements ofhealth, then we must devise new terminology for microbes that

improve growth performance or feed utilisation, microbes thatmodulate the gut microbiota without stimulating the immuneresponse (which may or may not provide a benefit by preventingubiquitous potential pathogens from colonising the digestive tract),microbes that improve gastric morphology or function and micro-organisms that prevent malformation or improve the quality of thefinal product. As there are no proposed definitions for such organisms,and for the sake of the current reviewwewill suppose that a probioticis any microbial cell provided via the diet or rearing water thatbenefits the host fish, fish farmer or fish consumer, which is achieved,in part at least, by improving the microbial balance of the fish. In thiscontext we regard direct benefits to the host as immuno-stimulation,improved disease resistance, reduced stress response, improved GImorphology etc and benefits to the fish farmer or consumer asimproved fish appetite, growth performance, feed utilisation,improvements of carcass quality, flesh quality and reducedmalformations.

3.1. Probiotic selection criteria

Previous reviews have proposed favourable characteristics for theselection of potential probionts for applications with fish species(Spanggaard et al., 2001; Balcázar et al., 2006a; Vine et al., 2006;Farzanfar, 2006; Gómez and Balcázar, 2008). Following on from thesepapers we propose an extended list of criteria for potential probionts,some of which are essential (E) and some considered as merelyfavourable (F). The more of these characteristics that are fulfilled by acandidate probiotic species, themore appropriate that species shall beconsidered and thus more likely to be an effective fish probiont: It

• must not be pathogenic, not only with regards to the host speciesbut also with regards to aquatic animals in general and humanconsumers (E)

• must be free of plasmid-encoded antibiotic resistance genes (E)• must be resistant to bile salts and low pH (E)• should be able to adhere to and/or grow well within intestinalmucus (F)

• should be able to colonise the intestinal epithelial surface (F)• should be registered for use as a feed additive (F)• should display advantageous growth characteristics (e.g. short lagperiod, a short doubling time and/or growth at host rearingtemperatures) (F)

• should exhibit antagonistic properties towards one or more keypathogens: in the case of salmonids one might focus on Aeromonassalmonicida, Vibrio (Listonella) anguillarum and Yersinia ruckeri (F)

• should produce relevant extracellular digestive enzymes (e.g.chitinase if chitin rich ingredients are to be incorporated into thediet or cellulase if the diet is rich in plant ingredients) or producevitamins. (F)

• should be indigenous to the host or the rearing environment (F)• should remain viable under normal storage conditions and robustenough to survive industrial processes (F)

As it is unlikely to find a candidate that will fulfil all of thesecharacteristics we should begin to further explore the possibilities ofsimultaneously using several probiotics or the use of probiotics withprebiotics (termed synbiotics) (Patterson and Burkholder, 2003).Through the combined application of multiple favourable probioticcandidates it may be possible to produce greater benefits (and satisfymore of the previously suggested characteristics) than the applicationofindividual probionts.

3.2. Mode of action and reported probiotic benefits in salmonids

The specific mode of action resulting in the observed host benefits isoften difficult to elucidate conclusively, due to thewide range of possiblemodes of action and the complicated synergistic multi-factorial

4 D.L. Merrifield et al. / Aquaculture 302 (2010) 1–18

relationships between them. Thus, much of our understanding of themechanisms behind probiotic effects stem from human studies.However, suggestedprobioticmodes of action infish include: productionof inhibitory compounds, competition for chemicals or available energy,competition for adhesion sites, inhibition of virulence gene expression ordisruption of quorum sensing, improvement of water quality, enhance-ment of the immune response, source of macro and/or micronutrientsand enzymatic contribution to digestion (Sugita et al., 1996, 1997; 1998;Vershuere et al., 2000; Olafsen, 2001; Vine, 2004; Gatesoupe 2008;Gòmez and Balcàzar, 2008; Ringø, 2008; Tinh et al., 2008). An extendeddiscussion of themechanisms of action of probiotic applications in fish ispresented by Tinh et al. (2008). Despite not always being fully aware ofthe precise mode of action it is clear that probiotic applications withsalmonids have resulted in elevated health status, improved diseaseresistance, growth performance, feed utilisation, carcass composition,gastric morphology, reduced malformations, GI colonisation and subse-quentlymicrobialmodulation. For a summaryof probiotic applications insalmonids refer to Table 1.

3.3. Immune response

Despite the large number of probiotic studies which have assessedimmunological and haematological parameters (Panigrahi et al., 2004,2005, 2007; Merrifield et al., 2010a,b,c; Kim and Austin, 2006a,b;Brunt et al., 2007, 2008; Irianto and Austin, 2002b; Balcázar et al.,2006a,b; Nikoskelainen et al., 2003; Raida et al., 2003; Arijo et al.,2008; Pieters et al., 2008) the immuno-modulatory effects ofprobiotics in fish systems are, at present, poorly understood. Probioticstrains of Carnobacterium have been demonstrated to augment innateimmune responses in rainbow trout, upon challenge with fishpathogens A. salmonicida and Y. ruckeri, increasing phagocytic activity,respiratory burst as well as serum and gut mucosal lysozyme activity(Kim and Austin, 2006a). A parallel study by the same group (Kim andAustin, 2006b), investigated the effects of the same probiotic strainson cytokine expression (IL-1β, IL-8, TNFα and TGFβ) by head kidney(HK) leukocytes and gut cells. None of the strains induced cytokineexpression in gut cells but augmented the expression of IL-1β andTNFα in HK leukocytes suggestive of a strengthening of innateimmunity in rainbow trout (Kim and Austin, 2006b). In a separatestudy by Panigrahi et al. (2007), three freeze-dried probionts(Lactobacillus rhamnosus, Enterococcus faecium and Bacillus subtilis)were fed to rainbow trout for 45 days at a density of 109 CFU g−1.Trout fed the probiotic supplemented feeds displayed enhancedsuperoxide anion production and serum alternative complementactivity. In addition, the expressions of IL-1 β, TNF and TGFβwere alsoupregulated in the spleen and HK. Such results are again suggestive ofaugmentation of innate immunity and possibly regulatory mechan-isms behind mucosal tolerance. The effects on the gut mucosal tissue,however, were not studied. Balcázar et al. (2007b) administereddietary strains of Lactococcus lactis, Lactobacillus sakei and Leuconostocmesenteroides to rainbow trout for 2 weeks at 106 CFU g−1. Comparedto the control group, HK leukocyte phagocytic activity and serumalternative complement activity were greater in probiotic fed fishgroups. In the case of L. sakei, superoxide anion production was alsoenhanced; however, no effect on lysozyme activity was detected.

The activity of serum lysozyme and alternative complement aretypical parameters assessed when investigating the effect of probio-tics on fish innate immune response (Panigrahi et al., 2004, 2005; Kimand Austin, 2006a,b; Balcázar et al., 2006a,b; Newaj-Fyzul et al., 2007;Merrifield et al., 2010a,b). Probiotics (Bacillus spp. and LAB) ofteninduce significant elevations of trout serum lysozyme activity (Newaj-Fyzul et al., 2007; Balcázar et al., 2007a; Merrifield et al., 2010a);however, the current findings are somewhat unclear as severalstudies have demonstrated variable effects of probiotics on serumlysozyme activity (Panigrahi et al., 2004, 2005; Balcázar et al., 2007a,b; Merrifield et al., 2010a,b).

With regards to probiotic effects on the adaptive immune system,Arijo et al. (2008) demonstrated that the administration of live probioticstrains resulted in the expressionof cross-reactive antibodieswhichwerespecific for outer membrane proteins and extracellular products ofbacterial pathogens, conferring a protective effect upon challenge withVibrio harveyi. In addition, the samegroup also demonstrated that dietaryprobiotics (Aeromonas sobria strain GC2) can protect rainbow troutagainst skin infections caused by the Aeromonas bestiarum (fin rot) andthe eukaryotic pathogen Ichthyophthirius multifiliis (white spot) (Pieterset al., 2008). This protection arose as a consequence of augmentation ofthe phagocytic response whereas a second protective probiotic strainenhanced respiratory burst activity (Pieters et al., 2008). A morecomprehensive overview of probiotics and disease resistance insalmonids is provided in the next section. From these studies, it wouldappear that probiotics enhance innate immunity and that the innatepathway response was dependent on the probiotic strain used.

In addition to direct effects, indirect effects of probiotics mayconfer health benefits. Fermentation products such as the short-chainfatty acid, butyrate, both modulate barrier function and regulateinflammatory processes, by decreasing epithelial permeabilitythrough up-regulation of tight junction proteins and suppression ofpro-inflammatory cytokines by induction of expression of anti-inflammatory, regulatory cytokines, respectively (van Nuenen et al.,2005). These short-chain fatty acids (SCFAs) are effectively acting asadopted regulators of both innate and adaptive immune mechanisms.

The current literature is indicative of probiotic dietary supple-ments effectively boosting innate immune defences and adaptiveimmune mechanisms of fish. So far, limited responses observing up-regulation of TGFβ and IL-10 expression are only suggestive of aprobiotic modulatory effect on maintenance of mucosal tolerance,crucial in tolerating the useful and mounting responses to thepathogenic. Thus, it would be fair to conclude that, similar to manycommercially available probiotic preparations in use for humans, thebest level of protection towards a broad spectrum of fish pathogenswill only be obtained through the use of probiotic dietary prepara-tions consisting of several probiotic strains, each strengthening aspecific area of the immune system.

3.4. Disease resistance

Both salmon and trout remain susceptible to a number of bacterialand viral diseases (these are summarised in Table 2 and are reviewedin detail by Groff and LaPatra (2000) and Toranzo et al. (2005)) as wellas a number of husbandry related stressors such as handling andtransportation that are of importance at different stages of growth andproduction.

3.4.1. Viral diseasesThe most severe viral diseases of salmonids are: infectious

pancreatic necrosis (IPN), viral haemopoietic septicemia (VHS),infectious haematopoietic necrosis (IHN), pancreas diseasse (PD),infectious salmon anaemia (ISA), heart and skeletal muscle inflam-mation (HSMI), and cardiomyopathy syndrome (CMS). Vaccination isthe traditional method for controlling such viral diseases but the levelof success is variable. For example, DNA vaccines against IHN and VHShave been developed and demonstrated to give protection againstdisease (Lorenzen et al., 2000; Kurath et al., 2006). However, despiteextensive vaccination of farmed Atlantic salmon against IPN inNorway (with some indications that vaccination may reduce losses)the efficacy of the vaccines in the field remains disputed (Wettenet al., 2007). Additionally, commercial vaccines against PD have beenin development and they have proved to be effective in some fieldtrials, but the duration of immunity has been questioned (McLoughlinand Graham, 2007). Furthermore, the causes of HSMI and CMS, whichhave been demonstrated to be infectious, are still under investigationand thus no vaccines are available (Poppe and Seierstad, 2003;

Table 1Summary of potential probiotic applications for salmonids.

Potential probiont (fish species) Parameters investigated References

Oncorhynchus mykissLa. lactis, Leu. Mesenteroides, L. sakei DR, IR, GM Balcázar et al. (2007b)a

Leu. mesenteroides, L. plantarum DR, GM, GP Vendrell et al. (2008)a

L. rhamnosus DR, GP Nikoskelainen et al. (2001)a

L. rhamnosus GM, IR Nikoskelainen et al. (2003)a

L. rhamnosus GM, IR Panigrahi et al. (2004)a

L. rhamnosus GM, IR Panigrahi et al. (2005)a

L. rhamnosus, B. subtilis, E. faecium IR Panigrahi et al. (2007)a,b

E. faecalis BC, GP, IR, DR Rodriguez-Estrada et al. (2009)a

B. subtilis DR, GM, IR Newaj-Fyzul et al. (2007)B. subtilis+B. licheniformis DR, IR Raida et al. (2003)a

B. subtilis+B. licheniformis BC, FU, GM, GP Bagerhi et al. (2008)a

B. subtilis+B. licheniformis, E. faecium BC, FU, GM, GP, IR Merrifield et al. (2009ab)a

B. subtilis+B. licheniformis, E. faecium, P. acidilactici GH, GM Merrifield et al. (2010d)a

Bacillus spp., A. sobria DR, IR Brunt et al. (2007)a

Bacillus spp., A. sobria IR Brunt et al. (2008)a

P. acidilactici BC, FU, GM, GP, IR Merrifield et al. (2009c)a

P. acidilactici, S. cerevisiae BC, FU, GM, GP, SM Aubin et al. (2005ab)a

P. acidilactici, S. cerevisiae DR, IR Quentel et al. (2004)a

C. divergens, C. maltaromaticum IR Kim and Austin (2006a)a

C. divergens, C. maltaromaticum IR Kim and Austin (2006b)b

C. inhibensa DR, GM Robertson et al. (2000)a

A. sobria DR, IR Brunt and Austin (2005)a,b

A. sobria, Brochothrix thermosphacta DR, IR Pieters et al. (2008)a

A. sobria DR, IR Arijo et al. (2008)a,b

A. hydrophila, V. fluvialis, Carnobacterium spp.,C. inhibens, V. alginolyticus and anunidentified Gram-positive coccus

DR, IR, GM Irianto and Austin (2002b)a

A. hydrophila, V. fluvialis, Carnobacterium spp.,and unidentified Gram-positive coccus

DR, IR Irianto and Austin (2003)a

Ps. fluorescens, Pseudomonas strains,Carnobacterium strains

DR Spanggaard et al. (2001)a,b

Ps. fluorescens DR Gram et al. (1999)a,b

S. cerevisiae,D. hansenii,R. glutinis GM Andlid et al. (1995)a

S. cerevisiae BE, GM Waché et al. (2006)a

Kocuria SM1 DR, IR Sharifuzzaman and Austin (2009)a

Enterobacter cloacae, Bacillus mojavensis DR, IR Capkin and Altinok (2009)a

Salmo truttaLa. lactis, Leu. Mesenteroides, L. sakei IR, GM Balcázar et al. (2007a)a

La. lactis, Leu. Mesenteroides, DR, IR Balcázar et al. (2009)a

Salmo salarC. inhibensa DR Robertson et al. (2000)a

C. divergens DR, GM, GP Gildberg et al. (1995)a

L. delbrueckii GH, DR, GM Salinas et al. (2008)a

C. divergens GH, DR, GM Ringø et al. (2007)b

Ps. fluorescens DR Gram et al. (2001)a,b

Ps. fluorescens DR Smith and Davey (1993)a

V. alginolyticus DR Austin et al. (1995)a,b

Genera abbreviations: A. = Aeromonas, B. = Bacillus, C. = Carnobacterium, D. = Debaryomyces, E. = Enterococcus, L. = Lactobacillus, La. = Lactococcus, Leu. = Leuconostoc, P. =Pediococcus, Ps. = Pseudomonas, R = Rhodotorula, S. = Saccharomyces, V. = Vibrio.Parameters investigated: BC — body composition, BE— brush border enzymes, DR— disease resistance, FU— feed utilisation, GH — gut histology, GM— gut microbiota (inclusive ofprobiont colonisation), GP — growth performance, IR — immunological/haematological response, SM — skeletal malformation.aIn vivo studies.bIn vitro experiments.cField experiments.1Carnobacterium strain K1 has been identified as Carnobacterium inhibens (Jöborn et al., 1999).

5D.L. Merrifield et al. / Aquaculture 302 (2010) 1–18

Kongtorp et al., 2004). An alternative approachmay include probioticsbut the potential for probiotic applications to improve resistanceagainst viral infections of fish are not presently known. If probioticscan elevate immune status, it is not unreasonable to speculate thatthis may reduce susceptibility to viral infections. Probiotics, such as L.rhamnosus, have been demonstrated to reduce rotavirus andpoliovirus infections in human patients (Marteau et al., 2001; deVrese et al., 2005). de Vrese et al. (2005) observed that L. rhamnosusincreased poliovirus neutralizing antibody titers (NT) and affected theformation of poliovirus-specific IgA and IgG in human patients.Moreover, feed supplementation with a Bacillus megeterium strain hasresulted in increased resistance to white spot syndrome virus (WSSV)in shrimp Litopenaeus vannamei (Li et al., 2009). Even if furtherresearch is necessary, the authors suggested that immuno-stimulation

is probably one of the important factors for the probiotic bacterium toenhance resistance of shrimp to WSSV.

3.4.2. Bacterial diseasesIt is generally accepted that in fish, bacterial pathogens can enter the

host byone ormore of threedifferent routes: (a) skin, (b) gills and (c)GItract (Lødemel et al., 2001; Ringø et al., 2004; Birkbeck and Ringø, 2005;Ringø et al., 2007a). If the GI tract is involved as an infection route,mucosal adhesion is considered to be a critical early phase in allinfections caused by pathogenic bacteria (Knudsen et al., 1999; Nambaet al., 2007).When the bacteria are able to colonise the intestinalmucus,they can cross the GI tract lining by transcellular or intracellular routes.

Extensive research has begun to demonstrate the efficacy of usingprobiotics to improve disease resistance and reduce mortalities of

Table 2Important viral and bacterial diseases of salmonid fish.

Disease Causative agent Treatment Species Reference

ViralInfectious pancreatic necrosis (IPN) IPN virus Vaccine S. salar Smail et al. (1992)Viral hemorrhagic septicemia (VHS) VHS virus DNA vaccine O. mykiss Lorenzen et al. (2000)Infectious hematopoietic necrosis (IHN) IHNvirus DNA vaccine O. mykiss Lorenzen and LaPatra (1999)Pancreas disease (PD) PD virus/salmonid alpha virus (SAV) Vaccine S. salar

O. mykissMcLoughlin and Graham (2007)

Infectious salmon anaemia (ISA) ISA virus Vaccine (Faroe Islands) S. salar Mjaaland et al. (1997)Heart and skeletal muscle inflammation (HSMI) HSMI virus No S. salar Kongtorp et al. (2004)Cardiomyopathy syndrome (CMS) CMS virus? No S. salar Ferguson et al. (1990)

BacterialFurunculosis Aeromonas salmonicida Vaccine S. salar

O. mykissToranzo et al. (2005)

Vibriosis Vibrio (Listonella) anguillarum Vaccine S. salarOncorhynchus sp.

Toranzo et al. (2005)

Cold water vibriosis Vibrio salmonicida Vaccine S. salar Bruno et al. (1986)Piscirickettsiosis Piscirickettsia salmonis No S. salar

Oncorhynchus sp.Mauel and Miller (2002)

Enteric redmouth disease (ERM)/yersiniosis Yersinia ruckeri Vaccine Oncorhynchus sp.Salmo sp.Salvelinus sp.

Furones et al. (1993)

Bacterial kidney disease Renibacterium salmoninarum No Oncorhynchus sp.S. salar

Evenden et al. (1993)

Winter ulcer Moritella viscosa Vaccine S. salarO. mykiss

Benediktsdottir et al. (2000)

6 D.L. Merrifield et al. / Aquaculture 302 (2010) 1–18

salmonids against bacterial diseases. Among the most importantbacterial diseases of salmonids are: furunculosis, vibriosis, cold watervibriosis, piscirickettsiosis, enteric redmouth disease (ERM) [akayersiniosis], bacterial kidney disease and winter ulcer (Table 2). Asummary of probiotic studies assessing disease resistance in salmo-nids is displayed in Table 3. These studies provide a solid foundation ofour knowledge regarding the probiotic potential to reduce aquacul-ture related diseases and it has been demonstrated that applicationsof viable, formalised, sonicated cells and cell-free supernatants arepotentially effective at reducing mortalities induced by a range ofbacterial pathogens (e.g. A. salmonicida, V./L. anguillarum, V. ordalii andLa. garvieae). However, we know very little regarding efficacy againstother important salmonid bacterial diseases such as V. salmonicida,Piscirickettsia salmonis, R. salmoninarum and Moritella viscosa. Futurestudies would do well to focus on these pathogens.

Although mineral oil-adjuvanted injection vaccines are by far themost efficient, giving rise to protection against diseases, the use ofthese vaccines often results in adverse side effects including extensiveadhesions and pigmentation of the peritoneum (Mutoloki et al.,2006). Therefore, alternative eco-friendly treatments must beconsidered. Treatments that may have a less significant environmen-tal impact include the strategic use of immuno-stimulants, probioticsand prebiotics.

3.4.3. Disease challenge methodsIn 2003, Ringø and co-authors put forward the statement “the

microbiologist must think as the military and use military strategy. Ifwe are going to defeat our enemies, the pathogens, we have to be atthe same place and time as them. If probiotic bacteria mostly colonisethe pyloric caeca, the probionts will have no effect if the pathogensmostly colonises the mid or hindgut regions and translocate in theseregions” (Ringø et al., 2003).

Based on this statement one might ask the question— how can weinvestigate the interactions between the beneficial bacteria vs. thepathogens in the GI tract of fish and observe the level of hostinfection? With this in mind we must consider that the potential ofprobiotics may be greater than some previous studies appear tosuggest. The intraperitoneal (IP) method of disease challenge over-rides one of the possible methods of probiotic protection against

pathogens by masking the potential effect of probiotic competitiveexclusion within the GI tract. Gastric probiotics may reduce or evenprevent gastric infection. IP challenges do not reflect the effect ofprobiotics on resistance to infection; rather they demonstrate theeffect of probiotics on infected fish. Therefore, it is recommended thatimmersion or cohabitation studies are conducted in future challengeexperiments in order to truly assess the full potential of candidateprobionts. The importance of theses potential antagonistic interac-tions in the gut are highlighted in a recent review, devoted to LAB vs.pathogens in the digestive tract of fish (Ringø et al. 2010a).

An additional aspect to consider in disease challenge studies is thefeeding duration with the probiotic prior to the challenge. Unfortu-nately, the effect of feeding duration on probiotic efficacy remainsscarcely investigated. Indeed in virtually all previously reportedstudies (Table 3), probiotics were administrated only for a few weeksprior to the challenge. To our knowledge, only one study in salmonidshas assessed the effect of long-term probiotic feeding prior to diseasechallenge (Quentel et al., 2004). This study, financially supported bythe OFIMER (Office National Interprofessionnel des Produits de la Meret de l'Aquaculture), investigated the efficacy of applying P. acidilacticiand Saccharomyces cerevisiae var. boulardii, alone or in combination,on the resistance of rainbow trout after intraperitoneal injection withY. ruckeri. The rainbow trout used in this experiment came from apreliminary study (Aubin et al., 2005b) where they were fed theprobionts from first feeding to 4 months of age. In the groups of fishfed diet supplemented with probiotic bacteria or yeast, a significantreduction in accumulative moralities was observed compared withfish fed commercial diet, at all challenge doses, thus demonstratingthat S. cerevisiae as well as P. acidilactici were found to significantlyincrease the protection of rainbow trout to experimental yersiniosis.Interestingly no production of antibody against Yersinia was detectedin any feeding groups, and thus the authors suggested that theprotection observed after the challenge may be consecutive of anactivation of the innate immune system.

3.5. Effect of probiotics on gut microbiota

In recent years, several reviews have suggested that the intestinalmicrobiota of fishmay play a role as a defensive barrier against enteric

Table3

Prob

ioticap

plications

forim

prov

ingdiseaseresistan

ceof

salm

onids.

Disea

seCa

usativeag

ent

Species

Challeng

emetho

dPo

tentialprob

iont

Redu

ced

mortalities

Notes

Referenc

es

Streptoc

occo

sis

S.iniae

O.m

ykiss

IPA.sob

ria

Yes

Live

cells

,disrupted

cells

andcellfree

supe

rnatan

teffective

Form

alised

cells

ineffective

Brun

tan

dAus

tin(2

005)

S.iniae

IPA.sob

ria,

Bacillu

ssp

p.Ye

sBrun

tet

al.(20

07)

Lactoc

occo

sis

La.g

arviea

eO.m

ykiss

IPA.sob

ria

Yes

Live

cells

,disrupted

cells

andcellfree

supe

rnatan

teffective

Form

alised

cells

ineffective

Brun

tan

dAus

tin(2

005)

La.g

arviea

eIP

A.sob

ria,

Bacillu

ssp

p.Ye

sBrun

tet

al.(20

07)

La.g

arviea

eCO

Leu.

mesen

teroides,L

.plantarum

Yes

Ven

drelle

tal.(20

08)

Furunc

ulosis

A.salmon

icida

O.m

ykiss

IPA.sob

ria,

Bacillu

ssp

p.Ye

sBrun

tet

al.(20

07)

A.salmon

icida

IM,C

O,IP

A.h

ydroph

ila,V

ibriosp

p.,C

arno

bacterium

spp.,

andan

uniden

tified

Gram-positiveco

ccus

Yes

Redu

cedfryan

dfing

erlin

gmortalities

Iriantoan

dAus

tin(2

002b

)

A.salmon

icida

COA.h

ydroph

ila,V

.fluv

ialis,C

arno

bacterium

spp.,

andan

uniden

tified

Gram-positiveco

ccus

Yes

Dea

dprob

ioticcells

used

Redu

cedfryan

dfing

erlin

gmortalities

Iriantoan

dAus

tin(2

003)

A.salmon

icida

COLa.lactis,Leu.

mesen

teroides

andL.sakei

Yes

Balcáz

aret

al.(20

07b)

A.salmon

icida

COL.rham

nosus

Yes

Nikoske

lainen

etal.(20

01)

A.salmon

icida

COC.

inhibe

nsa

Yes

Robe

rtsonet

al.(20

00)

A.salmon

icida

S.salar

COC.

inhibe

nsa

Yes

Robe

rtsonet

al.(20

00)

A.salmon

icida

COC.

divergen

sNo

Increa

sedmortalities

Gild

berg

etal.(19

95)

A.salmon

icida

COPs.fl

uorescen

sNo

Ps.fl

uorescen

sad

dedto

tank

water

Gram

etal.(20

01)

A.salmon

icida

–Ps.fl

uorescen

sYe

sPs.fl

uorescen

sad

dedto

tank

water

Smithan

dDav

ey(1

993)

A.salmon

icida

IMV.a

lginolyticus

Yes

Add

edto

tank

water

Aus

tinet

al.(19

95)

A.salmon

icida

invitro

C.divergen

sNA

Prev

entto

someex

tent

pathog

en-ind

uced

gutda

mag

eRing

øet

al.(20

07b)

A.salmon

icida

invitro

L.de

lbrueckii

NA

Prev

entto

someex

tent

pathog

en-ind

uced

gutda

mag

eSa

linas

etal.(20

08)

A.salmon

icida

S.trutta

Asymptom

atic

carrier

La.lactis,Leu.

mesen

teroides

Yes

Water

tempe

rature

increa

sedprog

ressivelyfrom

14°C

to16

°Cto

indu

cethedisease

Balcáz

aret

al.(20

09)

Vibriosis

V.a

nguilla

rum

O.m

ykiss

IMPseu

domon

assp

p.,C

arno

bacterium

spp.

Yes/no

Potentialp

robion

tsad

dedto

tank

water

Span

ggaa

rdet

al.(20

01)

V.a

nguilla

rum

IMPs.fl

uorescen

sYe

sPs.fl

uorescen

sad

dedto

tank

water

Gram

etal.(19

99)

V.a

nguilla

rum

IPA.sob

ria,

Bacillu

ssp

p.Ye

sBrun

tet

al.(20

07)

V.a

nguilla

rum

IPE.

faecalis

Yes

Redu

cedmortalitiesev

enfurthe

rwhe

nad

ministered

inco

njun

ctionwithdietaryMOS

Rodrigue

z-Estrad

aet

al.(20

09)

V.o

rdalii

IPA.sob

ria,

Bacillu

ssp

p.Ye

sBrun

tet

al.(20

07)

V.h

arveyi

IPA.sob

ria

Yes

Arijo

etal.(20

08)

V.a

nguilla

rum

S.salar

COC.

inhibe

nsa

No

Robe

rtsonet

al.(20

00)

V.a

nguilla

rum

IMV.a

lginolyticus

Yes

Add

edto

tank

water

Aus

tinet

al.(19

95)

V.a

nguilla

rum

invitro

C.divergen

sNA

Prev

entto

someex

tent

pathog

en-ind

uced

gutda

mag

eRing

øet

al.(20

07b)

V.o

rdalii

COC.

inhibe

nsa

Yes

Robe

rtsonet

al.(20

00)

V.o

rdalii

IMV.a

lginolyticus

Yes

Add

edto

tank

water

Aus

tinet

al.(19

95)

ERM

Y.ruckeri

O.m

ykiss

IPA.sob

ria,

Bacillu

ssp

p.Ye

sBrun

tet

al.(20

07)

Y.ruckeri

IPB.

subtilis+

B.liche

niform

isYe

sRa

idaet

al.(20

03)

Y.ruckeri

COC.

inhibe

nsa

Yes

Robe

rtsonet

al.(20

00)

Y.ruckeri

IMEn

teroba

cter

cloa

cae,

Bacillu

smojav

ensis

Yes

Capk

inan

dAltinok

(200

9)Y.

ruckeri

IPP.

acidila

ctici;S.

cerevisiae

var.bo

ulardii

Yes

Dietary

administrationof

theprob

ioticstrainsfor

4mon

thsbe

fore

thech

alleng

eAub

inet

al.(20

05b)

,Que

ntel

etal.(20

04)

Y.ruckeri

S.salar

COC.

inhibe

nsa

Yes

Robe

rtsonet

al.(20

00)

Finrot

A.b

estiarum

O.m

ykiss

IDA.sob

ria,

Brocho

thrixthermosph

acta

Yes

Pieterset

al.(20

08)

White

spot

Ichthy

ophthirius

multifiliis

O.m

ykiss

IMA.sob

ria,

Brocho

thrixthermosph

acta

Yes

Brocho

thrixthermosph

acta

noteffective

Pieterset

al.(20

08)

Other

Aerom

onas

sp.

O.m

ykiss

IPB.

subtilis

Yes

Viable,

form

alised

,disrupted

cells

andcell-free

supe

rnatan

teffective

New

aj-Fyz

ulet

al.(20

07)

Bacterialg

eneraab

brev

iation

s:A.=

Aerom

onas,B

.=Ba

cillu

s,C.

=Ca

rnob

acterium

,L.=

Lactob

acillus,L

a.=

Lactococcu

s,Leu.

=Leuc

onostoc,Ps.=

Pseu

domon

as,S

.=Streptococcu

s,V.=

Vibrio.

Challeng

emetho

dab

brev

iation

s:IP

=intrap

eriton

ealinjection

,ID

=intrad

ermal

injection,

IM=

immersion

,CO

=co

habitation

,NA=

notap

plicab

le.

aCa

rnob

acterium

strain

K1ha

sbe

eniden

tified

asCa

rnob

acterium

inhibe

ns(Jöb

ornet

al.,19

99).

7D.L. Merrifield et al. / Aquaculture 302 (2010) 1–18

Table 4Summary of probionts that have been recovered from the digestive tract of salmonidsafter feeding on supplemented diets.

Probiont Effect on indigenousmicrobiota assessed?a

Reference

Oncorhynchus mykissVibrio alginolyticus No Irianto and Austin (2002b)Vibrio fluvialis No Irianto and Austin (2002b)Aeromonashydrophila

No Irianto and Austin (2002b)

B. subtilis No Newaj-Fyzul et al. (2007)B. subtilis+B. licheniformis

Yes Bagheri et al. (2008)No Merrifield et al. (2009abd)

C .inhibens No Irianto and Austin (2002b)Robertson et al. (2000)

Carnobacterium spp. No Irianto and Austin (2002b)C. maltaromaticum No Kim and Austin (2006a)Carnobacteriumdivergens

Kim and Austin (2006a)

P. acidilactici Yes Aubin et al. (2005a,b)No Merrifield et al. (2009c,d)

S. cerevisiae Yes Aubin et al. (2005a)Yes Waché et al. (2006)

Leu. mesenteroides No Balcázar et al. (2007b)No Vendrell et al. (2008)

La. lactis No Balcázar et al. (2007b)

8 D.L. Merrifield et al. / Aquaculture 302 (2010) 1–18

infections (Birkbeck and Ringø, 2005; Ringø et al., 2005; Gómez andBalcázar, 2008; Ringø and Olsen, 2008; Ringø et al., 2010a); asprobiotics induce host benefits, in part at least, by modulation of thehost microbiota it is pertinent to discuss the effect of probiotics on thegut microbiota of salmonids. The effect of probiotics on the gutmicrobiota of fish has been discussed in detail previously (Ringø et al.,2005) but an update of literature is required. A wide range ofprobionts have been recovered from the digestive tract of salmonidsafter feeding on supplemented diets; these include Aeromonas spp.,Bacillus spp., Carnobacterium spp., Enterococcus spp., Lactobacillus spp.,Lactococcus spp., Leuconostoc spp., P. acidilactici, Saccharomyces spp.and Vibrio spp. (refer to Table 4).

The continual application of bacterial cells (LAB, Bacillus spp. andcertain Gram-negative spp.) to salmonids may lead to high levels ofcolonisation and modulated GI microbial populations (Irianto andAustin, 2002b; Panigrahi et al., 2004, 2005; Aubin et al., 2005a; Kimand Austin, 2006a; Balcázar et al., 2007a,b; Bagheri et al., 2008;Merrifield et al., 2010a,b,c,d). Routinely, investigators enumerateprobiont and total viable levels of the gut microbiota after feedingon supplemented diets but unfortunately few studies assess thecomposition of the indigenous microbiota. The main exceptions insalmonids are Gildberg et al. (1995), Aubin et al. (2005a,b), Wachéet al. (2006) and Bagheri et al. (2008). Despite providing useful dataand demonstrating broad level alterations on the gut microbiotaprevious studies do not present the full picture due to the culture-dependent methods used. A plethora of culture-independentmethods are available based on the sequence variability of the 16Sand 23S rRNA genes that allow us to identify and quantify intestinalmicrobiota. Typically, when assessing the gut microbiota of fishDGGE remains the method of choice due to its rapid and inexpensivenature (Liu et al., 2008, Dimitroglou et al., 2009; Zhou et al., 2007,2009). Indeed, Kim and Austin (2006a) demonstrated the usefulnessof DGGE to assess the gastric longevity of carnobacteria strains fedto rainbow trout after reverting to non-supplemented diets. DGGEfingerprints revealed that Carnobacterium maltaromaticum andCarnobacterium divergens could survive in trout intestine for atleast 3 weeks after dietary supplementation ended.

The current literature provides a foundation, but our knowledgeof the effect of probiotics on the gut microbiota of salmonids islimited because [1] most studies tend to be restricted to theenumeration of the probionts without investigation of theindigenous microbiota, [2] modern culture-independent molecularbased techniques are not frequently utilised and [3] data isprimarily restricted to rainbow trout. Future probiotic studiesshould incorporate PCR-DGGE techniques with subsequent 16SrRNA sequence analysis and/or microbial community diversityanalysis, nMDS and similarity indices which have been used tostudy the gut microbiota of fish previously (Dimitroglou et al.,2009; Merrifield et al., 2009). Furthermore, quantitative culture-independent methods, such as FISH and qRT-PCR, should become aroutine method for future investigations.

L. sakei No Balcázar et al. (2007b)L. plantarum No Vendrell et al. (2008)L. rhamnosus No Nikoskelainen et al. (2003)

No Panigrahi et al. (2005, 2005)E. faecium No Merrifield et al. (2009abc)Salmo salarCarnobacteriumdivergens

Yes Gildberg et al. (1995)

C. inhibensb No Jöborn et al. (1997)No Robertson et al. (2000)

Salmo truttaLeu. mesenteroides Yes Balcázar et al. (2007a)La. lactis Yes Balcázar et al. (2007a)L. sakei Yes Balcázar et al. (2007a)

a Enumeration of TVC of indigenous levels or probiotics cells = No; identification ofindigenous genera/species affected and/or use of alternative molecular methods forassessing changes in diversity, richness etc = Yes.

b Carnobacterium strain K1 was later identified as C. inhibens.

3.6. Gastrointestinal morphology

The endogenous gut microbiota influence the development ofthe gut and are key components involved in the regulation ofmucosal tolerance, development and differentiation (Rawls et al.,2004, 2007; Bates et al., 2006); however, maintenance of a healthygut microbiota is likely to beneficially affect the gut epithelialarchitecture at both the developmental and post developmentalstage. Reducing the number of potential pathogens present withinthe GI tract may reduce mucosal damage and lead to improvedabsorptive surface area. Indeed, many fish pathogens can disrupt theintegrity of the intestinal epithelium (Ringø et al., 2007a,b; Salinaset al., 2008; Ringø et al., 2010a).

The histological effect of probiotics on the gut epithelium ofsalmonids has been assessed in vitro (Løvmo, 2007; Ringø et al.,2007b; Salinas et al., 2008; Ringø et al., 2010a). For example, Ringø et al.(2007b) exposed the Atlantic salmon foregut to C. divergens, originallyisolated from distal intestine of Atlantic salmon, as well as two wellknown pathogens: A. salmonicida and V. (L.) anguillarum. Light andelectron microscopy demonstrated that pathogen-induced damage tothe Atlantic salmon foregut could not be prevented or reversed, butcould be marginally reduced in some cases.

Despite such in vitro studies, in vivo data regarding the effectsof probiotics on the intestinal morphology of salmonids is scarce.To the authors' knowledge, the only probiotic study which hasdemonstrated in vivo that probiotics may enhance the intestinalmicrovilli morphology of salmonids is that of Merrifield et al.(2010d). Merrifield and colleagues demonstrated in a preliminarystudy that dietary applications of P. acidilactici could significantlyimprove microvilli length of the rainbow trout proximal intestinecompared to the control group. However, microvilli density wasnot affected. The study also demonstrated that dietary treatmentsof B. subtilis+B licheniformis and E. faecium did not affectmicrovilli length or density. The reason for the lack of benefits inthe B. subtilis+B licheniformis and E. faecium was not elucidatedbut the authors speculated that it may be due to the perceived lackof mucosal colonisation (no colonisation by probiont-like cells)observed by electron microscopy in these groups. Instead, cellsappeared to colonise the mucus layer. Further study is required to

9D.L. Merrifield et al. / Aquaculture 302 (2010) 1–18

understand these observations and assess the level of host-microbe interaction regarding the probionts investigated.

3.7. Nutrition

Although many now consider probiotic applications in terms ofhealth we must also remember that modulation of gut microbiotaand enhanced gut morphology may help to improve the nutritionof the host. Modulation of the gut microbiota may enhanceenzymatic activity (Waché et al., 2006) and the production ofvitamins which are likely to bolster health and nutrition in thehost. Indeed numerous studies have shown that the application ofprobiotics can improve feed conversion, growth rates and weightgain of fish including salmonids (e.g. Bogut et al., 1998, 2000;Taoka et al., 2006a; Wang et al., 2008b; Bagheri et al., 2008; ). Forexample, Bagheri et al. (2008) demonstrated that the application ofB. subtilis+B. licheniformis could significantly improve rainbowtrout fry feed conversion ratio (FCR), specific growth rate (SGR),weight gain and protein efficiency ratio (PER) after 2 monthsfeeding on diets containing 3.8×109 CFU g−1. Furthermore, com-pared to the control group body protein composition was elevatedand lipid levels decreased in the probiotic group. It is recom-mended that future studies monitor growth parameters and bodycomposition.

Beyond these parameters, little data exist with regards to othercontributions to the digestive function. However, work by Aubin et al.(2005a) revealed that dietary P. acidilactici alleviated vertebral columncompression syndrome (VCCS) in rainbow trout to a similar extent asantibiotics. The authors postulated that this confirmed that infectionwas the root cause of the syndrome and that probiotics were able toreduce disease prevalence, by either competing with gut pathogens orby stimulating favourable conditions in the intestine. This is a reasonablehypothesis, but in light of recent studies (Merrifield et al., 2010c,d), wehypothesise that P. acidilactici may improve bone formation/mineral-ization via improved mineral uptake. Merrifield et al. (2010c) observeda significantly reduced condition-factor in rainbow trout fed P.acidilactici versus the control group; effectively increasing the lengthto weight ratio. Given that no other growth parameters had beenaffected in that study, it is clear that trout receiving probiotic treatmentswere in some way elongated or that the control group were to someextent stunted. The proposedmode of action of probiotics acidifying thegastric environment may have played a significant part in thesolubilization and thus uptake of minerals from the digestive tract.Indeed, it is known that acidification, leading to reduction and thushigher solubility of minerals, increases the uptake of many metal ions.This is most notably demonstrated as ascorbic acid reduces iron to itsmore soluble form: the reason that vitamin C is associatedwith a higheriron uptake efficiency (Fairweather-Tait, 1995). In the case of probiotictreatments, this acidification is achieved through the production oflactic acid and short-chain fatty acids. Evidence in higher vertebratessupports this suggestion: in humans, low serum values of magnesium,copper and zinc are associated with osteoporosis in post-menopausalwomen (Scholz-Ahrens et al., 2007), a clear correlation of mineralassimilation/circulation and correct bone formation. In their review,Scholz-Ahrens et al. (2007) also highlighted rat-studies showing thatprebiotics stimulated the absorption of iron and of bone-relevantminerals such as calcium, magnesium and zinc. Few studies haveassessed the effects of probiotics or synbiotics on metabolism ofminerals or subsequent effects on bonehealth; yet it has been suggestedthat there is an independent probiotic effect on facilitating mineralabsorption (Scholz-Ahrens et al., 2007).

Using transmission electron microscopy (TEM) Merrifield et al.(2010d) observed increased enterocyte endocytic activity andincreased microvilli length in rainbow trout fed dietary P. acidilactici.This may support the present hypothesis that an increased uptake ofminerals/calcium may occur in trout fed P. acidilactici. However,

further studies are required to confirm this speculative hypothesis.There appears to be a growing volume of literature demonstrating thebeneficial role of probiotics in the uptake ofminerals from the gut, andsubsequent deposition in the bone. However, a further possibility alsoexists to explain how probiotics can improve mineral uptake from theintestinal lumen. It has been proposed that the bacterial activity in thegut is able to degrade the mineral-complexing phytate (Scholz-Ahrens et al., 2007). Whilst aquaculture diets tend to be supplemen-ted with phosphorus to ensure that adequate free-phosphorus isavailable in the ration, little attention is paid to the impact of phyticacid on other minerals. In order to further explore this avenue, it isproposed that trials be conducted looking at the impact ofsupplemental phytase enzyme on the bone development in farmedsalmonids.

3.8. Feeding strategies

Many studies have now provided proof of concept that probioticscan be effective for use within aquaculture but probiotic applicationmethods have varied greatly and are not always practical forproduction level fish farming. If a salmonid fish farmer decides touse probiotics how should the application be conducted? We mustconsider: [1] the probiont, [2] supplementation form, [3] vector ofadministration, [4] dosage level and [5] duration of application.

3.8.1. Choosing a probiontBacillus spp., LAB, certan Gram-negative spp. and yeast have all

demonstrated benefits for salmonids (refer to Table 1). Initially, it wassuggested that probiotic bacteria could be host-specific with theireffects limited to their natural hosts (Fuller, 1973; Mäyrä-Mäkinen etal., 1983) but later studies allow us to question whether this is true(Gildberg and Mikkelsen, 1998; Rinkinen et al., 2003; Salinas et al.,2008). In either case, the specific probiont will likely be dependent onthe fish species, rearing conditions and desired outcome of supple-mentation (i.e. immuno-stimulation, disease prevention, improvedgrowth performance etc).

3.8.2. Supplementation formMost commonly in salmonid studies, live-cultures are sprayed or

top-dressed onto basal diets (e.g. Panigrahi et al., 2005, Balcázar et al.,2007a,b; Vendrell et al., 2008; Merrifield et al., 2010a,b,c,d) butfreeze-dried/lyophilised cells (e.g. Aubin et al., 2005a; Panigrahi et al.,2005, 2007; Merrifield et al., 2010c), dead cells (e.g. Irianto andAustin, 2003; Panigrahi et al., 2005; Newaj-Fyzul et al., 2007),disrupted cells (e.g. Brunt and Austin, 2005; Newaj-Fyzul et al.,2007), cell-free supernatants (e.g. Brunt and Austin, 2005; Newaj-Fyzul et al., 2007) and spores (e.g. Raida et al., 2003; Bagheri et al.,2008) have all showed some degree of success. With regards toapplications at the farm level it may be more practical to use deadcells, lyophilised cells or spores rather than culturing live cells. Fewstudies have compared both live and dead probiotic applications infish but studies have demonstrated that probiotic viability can be afactor (Brunt and Austin, 2005; Panigrahi et al., 2005; Taoka et al.,2006b). Panigrahi et al. (2005) concluded from their study, that thepotential benefits of heat-killed cells should not be overlooked butthat viable forms induced better results. Furthermore, as nodifferences between the live-sprayed and freeze-dried forms wereobserved in the investigation, Panigrahi et al. (2005) suggested thefreeze-dried administration method merited further consideration asa practical mode of delivery for aquaculture practices. One of thebeneficial attributes of Bacillus species for applications as probiotics istheir spore-forming abilities which allows for greater viability afterpelleting and high resistance to gastric conditions (Hyronimus et al.,2000; Casula and Cutting, 2002; Hong et al., 2005). Furthermore, theapplication of dietary Bacillus spores has proved effective in probioticapplications for trout (Raida et al., 2003; Bagheri et al., 2008). As it is

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not practical to mass culture cells prior to application at the farmperhaps lyophilised cells or spores may be a more practical option.

3.8.3. Vector of administrationMost commonly probiotics have been administered via the feed;

however, some information regarding administration of probiotics tosalmonids via rearing water is available. Such application has provedboth effective and ineffective at reducingmortalities (refer to Table 3).There is limited data comparing the effectiveness of water basedsupplementation compared to oral administration in salmonids, butwater based applications are likely to be limited in flow-throughrearing systems and sea/lake cages. These methods may be moreapplicable in re-circulation facilities or for bathing treatments appliedregularly or during times of disease. Furthermore, data regardingwater-administered probionts to salmonids appear to be availableonly with regards to disease resistance, which leaves many questionsregarding the efficacy to other host related parameters.

3.8.4. Dosage levelDose can be important as differences of host response between

different dietary probiotic levels have been observed (Nikoskelainenet al., 2001, 2003; Panigrahi et al., 2004; Bagheri et al., 2008).Panigrahi et al. (2004) investigated the potential dose–responserelationship of rainbow trout with L. rhamnosus. Panigrahi et al.(2004) assessed the cellular and humoral immune response ofrainbow trout fed diets containing L. rhamnosus at either 109 or1011 CFU g−1 for 30 days. After 30 days feeding, both probiotic groupsdisplayed significantly higher HK leukocyte phagocytic activity butonly the high level group displayed significantly improved serumlysozyme and alternative complement activity compared to thecontrol group. High LAB levels were recovered from the GI tractthroughout the supplemented feeding period but LAB levels in theintestine also appeared to be dose dependent.

Dose dependent studies are currently limited and somewhatcontradictory. Further studies are required, in particular with salmon,before guideline levels can be suggested with any degree ofconfidence. Appropriate levels are likely to vary depending on theprobiont species, host fish species, host physiological status, rearingconditions and the specific goal of the feeding application (i.e. health,disease resistance, nutrition etc).

3.8.5. Supplementation durationStudies have assessed potential probiotic applications for periods

as short as 6 days (Jöborn et al., 1997) and for periods of up to5 months (Aubin et al., 2005a). In some rare cases, the influence ofprobiotic feeding duration on treatment efficacy has been studied(Panigrahi et al., 2005; Balcázar et al., 2007a; Sharifuzzaman andAustin, 2009). However, information on long-term efficacy is notavailable. Supplementation has proved to provide short-term benefitsbut generally probionts have not been detected within the GI tract forperiods beyond one to three weeks after reverting to non-supple-mented diets (Robertson et al., 2000; Balcázar et al., 2007a; Panigrahiet al., 2005; Kim and Austin, 2006a) and presumably probioticbenefits are lost after the probiont is removed from the host.Therefore there appear to be 3 distinct options for administrativestrategy: [i] short-term application confined to times of need, [ii]constant supplemented feeding or [iii] cyclic feed of supplementeddiets.

[i] Short-term supplementation has proved to be effective atgastric colonisation, stimulating the immune system and providingprotection against disease when fed prior to pathogenic challenge/infection (Irianto and Austin, 2002b, 2003; Brunt and Austin, 2005;Brunt et al., 2007; Newaj-Fyzul et al., 2007; Pieters et al., 2008). Suchstudies provide important proof of concept that the use of selectedprobiotics can be an effective method for protection against disease;however, a fish farmer cannot predict when the onset of disease may

occur in order to provide probiotic feeding in the weeks prior toinfection. Will treatment be effective after the onset of disease hasbeen detected? Further work is required to answer this question.Restricted feeding to times conducive of stress may be beneficial yetcurrently there is little data to support this strategy.

[ii] Constant supply of probiotics, incorporated into the diet as a feedsupplement,mayprovide benefits but there is little data available for thelong-term use of probiotics. However, the study by Aubin et al. (2005a)provides someuseful data regarding long-term applications. Aubin et al.(2005a) compared probiotic recovery levels over time and observedthat levels were higher after 20 days (with P. acidilactici levels of log2.5 CFU g−1 and S. cerevisiae of log 4.5 CFU g−1) than after 5 months (P.acidilactici levels of log 0.9 CFU g−1 and S. cerevisiaewere not detected).When considering long-term use of probiotics it is worth consideringthat the immune response is often diminished with long-term use ofimmuno-stimulants which often leads to the immune status revertingback to control levels or in extreme cases may lead to immuno-suppression (Sakai, 1999; Smith et al., 2003;Bricknell andDalmo, 2005).Although it is not currently clearwhether this is the casewith long-termapplication of probiotics, we must consider the possibility that it maynot bepertinent touse constantprobiotic supplementation for extendedperiods.

[iii] Short-term-cyclic (termed pulse-administration by Bricknelland Dalmo, 2005) probiotic feeding strategies may be beneficial, as isthe case of some immuno-stimulant products, although there is nodata currently available to support or disprove this hypothesis. Such astrategy could involve feeding probiotic supplemented diets and un-supplemented diets alternately for short periods (e.g. for periods of 2or 4 weeks) cyclically. Application in this way may provide the directbenefits of short-term application during the supplemental feedingphase and during the un-supplemented stage where gastric probioticpopulations persist for a number of weeks (Balcázar et al., 2007a; Kimand Austin, 2006a) may provide a level of protection against transientpathogens and could continue to induce some degree of immuno-stimulation (Nikoskelainen et al., 2003; Balcázar et al., 2007a). Thisstrategy may help to avoid over-stimulating the immune responsewhilst maintaining a level of protection/immuno-stimulation. Cur-rently there is no data to support this hypothesis and future researchshould consider this application strategy.

Current research provides proof of the probiotic concept forsalmonids but applications within these studies are so varied andoften impractical for industrial scale farming that it is difficult to planan effective feeding strategy for commercial level applications. Futurework, including practical evaluation under farming conditions, oncesafety concerns have been considered, should focus on providing aremedy to this.

3.9. Issues with industrial level scale-up

Following on from the aforementioned problems and lack ofknowledge pertaining to applications of feeding strategies mentionedin the previous section, several other issues have, and continue to,hamper the transition from scientific trials to industrial scaleapplications. Several studies have been conducted at fish farms(refer to Table 1) but industrial level applications on mass are notfrequently reported. It is likely however, that industrial scaleapplications are relatively common, as commercial bodies wish toassess their products but due to issues regarding intellectual propertyrights such data is confined to industrial stakeholders and not widelyshared with the academic community until sometime later.

It is important to mention that in all cases, at least within theEuropean Union, each field trial involving feed additives not yetauthorised require approvals by national food safety associationequivalents; making industrial level applications on mass difficult.From industrial and commercial perspectives, such trials are of courseessential to demonstrate the efficacy of probiotics at the farm level

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and to evaluate the financial outcome of using such products in thefield. Furthermore these trials also demonstrate the feasibility of theprobiotic application form a practical point of view (conditions of use,stability under different storage conditions etc). Indeed, in order to beused successfully at an industrial scale, technological issuesconcerning probiotic incorporation into extruded feeds must beconsidered because as discussed previously, in many cases probioticviability impacts effectiveness. Thus probiotic microorganisms mustsurvive the stressful conditions of feed processing and storage whichis a particular concern in aquaculture since aquafeed processingconditions are physically intensive compared to terrestrial animalfeeds. Therefore, probiotic incorporation in aquafeeds remains limitedat industrial levels, particularly due to the drawback of heat stability.In order to overcome such issues, novel approaches in application arerequired. Providing probiotic living cells in a protected form with aphysical barrier against adverse environmental conditions is atechnique currently receiving considerable interest. For instance,microencapsulation of probiotic bacterial cells has been developed(Kailasapathy, 2002) and patented microencapsulated bacteria oryeasts are today available on the market (Ziggers, 2005). Recentlydramatic improvement in this technology has been achieved by yeastand bacteria producing companies and some active yeast commercialproducts have even been reported to resist temperatures up to 90 °C(M. Castex, pers. communication). However, the current microencap-sulation technology is not yet sufficient to protect live bacteria in thecase of extruded feeds. While developing new microencapsulationtechnologies, the only alternative for now is to develop alternativesolutions with the feed manufacturers in order to apply probiotics ontheir feed products. Several systems exist and the post-pelletingspraying in oil or water is generally the best option. For instance thisprocedure applied to salmon feeds has shown efficient results interms of homogeneity and conformity to the expected dosage at bothlaboratory and industrial levels (Ziggers, 2005). Furthermore, afterfollowing the required procedure for pressure, temperature, flow rateetc, probiotic survival remains stable on pellets for a few months.However, the implementation of post-pelleting systems on feedplants generally requires significant investments, including specificequipments such as screw coater and probiotic injection units, andcan only be applied after the efficacy of the concerned probiotic hasbeen fully validated in the field. In the future of probiotic developmentat an industrial scale, alliance and transversal experiences betweenbiological technology and industrial processing must be conducted inorder to offer innovative and easy to use solutions for the feedindustry.

Finally, one of the main stumbling blocks from a practical point ofview, in order to be used at a large scale, is that candidate probioticspecies must satisfy stringent regulations. Indeed, safety considerationsare increasingly taken into account for the development of probiotics. Asan example, the European Union regulates the authorisation, marketingand use of probiotics as feed additives under the European Parliamentand Council Regulation (EC) No (1831). In accordance with thisRegulation, the Commission, having first consulted the European FoodSafety Authority (EFSA), has established rules (Commission Regulation(EC)No 429/ (2008)) concerning the preparation and the presentation ofapplications. Then the registration of a probiotic requires the preparationof adossierwithdata and studies demonstrating efficacy and safetyof theproduct for animals, consumer andenvironment. This safety concerns areof utmost importance in aquaculture since, as mentioned byWang et al.(2008b), new species and specific strains identified as potentialprobiotics for aquatic species do not share the historical safety oftraditional orwidely tested strains suchas LABs (Adams, 1999).However,a long-term Research & Development project, initiated from 2001through the OFIMER program with BioMar and several researchinstitutions, has resulted in the first EU-approval of the use of probioticsfor salmonids in August 2009 (http://www.biomar.com/es/Corporate/News/First-feed-with-a-probiotic-/). This has allowed BioMar to develop

an innovative dietary probiotic concept andwill result in the introductionof the first approved industrial trout and salmon feed containingprobiotics in 2010 (M. Autin, pers. communication).

3.10. Future probiotic work

Scientists working on the topic of probiotics should also focus theirresearch on the use of other molecular methods than DGGE. As littleinformation is available on quorum sensing, different stainingmethods (e.g. immunogold, FISH and green fluorescent proteinlabelled bacteria) should be utilised to investigate adherence andcolonisation of probiotic and pathogenic bacteria and their interac-tions within the fish digestive tract. Scientists might profitablyinvestigate the interactions between probiotics and pathogens inthe digestive tract of fish as suggested by Ringø et al. (2010a). Inaddition to molecular techniques to evaluate the microbial commu-nity as a result of probiotic supplementation, important endogenousbacteria isolated by traditional cultivation should be investigated fortheir metabolic capabilities such as degradation of anti-nutrients, as ithas been shown that LAB fermentation can improve the nutritionalvalue of soybean meal (Refstie et al., 2005). This is highly relevant toinvestigate as one of the main challenges in aquaculture is the use ofplant protein sources in the diets (Gatlin et al., 2007). Moreover,adhesion is one of the most important selection criteria for probioticbacteria because it is considered a prerequisite for colonisation.Although some information is available regarding probiotic adhesionto and growth within fish intestinal mucus, it is essential thatprobiont–mucosal interactions be assessed in future studies. It is atpresent not clear what role the gut microbiota, or indeed probiotics,play in mediating salmonid gastric development, gene expression andmucosal tolerance. Thus, much of our understanding regarding effectsin salmonids, and fish in the broad sense, come from studies withhigher vertebrates. However, Kim and Austin (2006b) assessed theeffects of Carnobacterium on gene expression of gut cells and HKleukocytes in vitro by co-culturing with the probiotic cells andconcluded that although the probiotics did not significantly inducecytokine mRNA in gut cells, the probiotics could stimulate innateimmunity as demonstrated by the expression ratios of IL-1β and TNFαof HK cells. The study provides a platform for our understanding of theeffects of probiotics on the expression of cytokines in gut cells butfurther in vivo work is required to provide a more broad understand-ing of the complex interactions of probiotics and the host mucosa.Future work should aim to enhance our understanding of theseinteractions by isolating gut cells of probiotic fed salmonids andassessing the expression of immune activatory or immunoregulatorycytokines (e.g. IL-1, IL-8 and TNFα and INFγ), pattern recognitionreceptors (eg. TLRs and scavenger receptors) and anti-microbialproteins (e.g. IRF3, INFα type 1, Mx 1, TLR3, TLR7, TLR9, NOS2 andGBP). Such studies will elucidate the cellular and molecular mechan-isms involved in probioticmodulation of themucosal immune system,informing future approaches in aquaculture in the maintenance of anefficient immune system which is reflected in fish quality andproductivity.

4. Prebiotics

The use of probiotics in many cases, as discussed previously, maybe difficult in commercial aquaculture because of the low viability ofthe bacteria after pelleting and during storage, leaching from the feedparticle in rearing water, as well as problems related with feedhandling and preparation. As an alternative (or also considered for usein tandem: synbiotics), prebiotics have been assessed in an attempt toovercome issues associated with probiotic applications. From anendothermic point of view, a prebiotic is defined as a non-digestiblefood ingredient that beneficially effects the host by selectivelystimulating the growth and/or the activity of specific health

Table 5Summary of potential prebiotic applications for salmonids.

Species Prebiotic Parametersinvestigated

References

Oncorhynchusmykiss

MOS GM, GH Dimitroglou et al. (2009)c

MOS GP, FU, GH Yilmaz et al. (2007)a

MOS GP, FU, IR Staykov et al. (2007)a

MOS BC, GP, IR, DR Rodriguez-Estrada et al.(2009)a

GroBiotic A* BC, DR, GP, IR Sealey et al. (2007)a

Salmo salar MOS GP, FU, GH, RP Dimitroglou et al.(unpublished data)c

MOS, GOS,FOS

GP, FU, BC Grisdale-Helland et al. (2008)a

Inulin DE, OI, PC, GH Refstie et al. (2006)a

Inulin BP, OI, GM, GH Bakke-McKellep et al. (2007)a

Salvelinusalpinus

Inulin GH Olsen et al. (2001)a

Inulin GM Ringø et al. (2006)a

Parameters investigated: BC— body composition, DE— digestive enzyme activity, DR—

disease resistance against V. (L.) anguillarum, FU — feed utilisation, GH — gut histology,GM — gut microbiota, GP — growth performance, IR — immune response, RP —

resistance to parasitic infection, SM — skeletal malformation, OI — organosomaticindices, PC — plasma chemistry, BP — body parameters.aIn vivo studies.bIn vitro experiments.cField experiments.*GroBiotic®-A is a mixture of partially autolyzed brewers yeast, dairy ingredientcomponents and dried fermentation products.

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promoting bacteria that can improve the host health (Gibson andRoberfroid, 1995).

Prebiotics mainly consist of oligosaccharides promoting beneficialbacterial growth within the GI tract (Yazawa et al., 1978; Gibson et al.,2003). Readers with specific interest into the classification and types ofcarbohydrates with potential to be classed as prebiotics are referred tothe reviews of Gibson et al. (2004) and Roberfroid (2007). In a review forhuman intestinalmicrobiota theprebioticdefinitionwasupdated.Gibsonet al. (2004) suggested that a prebiotic has to:

• resist gastric acidity, hydrolysis by (mammalian) enzymes and GIabsorption.

• be fermented by the intestinal microbiota.• stimulate selectively the growth and/or activity of intestinal bacteriaassociated with health and well-being.

According to the authors, each one of these criteria is importantbut the third criterion, concerning the selective stimulation of growthand/or activity of beneficial bacteria, is the most important and themost difficult to achieve. This is especially true because this steprequires anaerobic sampling and reliable quantitative microbialanalysis should be undertaken for a wide range of bacterial species(i.e. total aerobic and anaerobic bacteria as well as total counts ofBifidobacteria, Enterobacteria, Lactobacillus, etc). According to Gibsonet al. (2004), only three oligosaccharides were classified as prebiotics:inulin, transgalactooligosaccharide (TOS) and lactulose. Amore recentstudy includes fructooligosaccharides (FOS) in the list of prebiotics(Roberfroid, 2007). Due to the limited data available regarding fish,the carbohydrates that have fulfilled the requirements, or have shownpotential to fulfil the requirements, of the definition of prebiotic in theaforementioned reviews will be discussed in context to salmonids.Prebiotics have demonstrated some benefits in fish (Burr et al., 2005;Gatlin et al., 2006; Ringø and Olsen, 2008; Ringø et al., 2010b) but theuse of prebiotics in salmonid studies remains relatively limited(Table 5). Based on the prebiotic results available per se, Ringø et al.(2010b) concluded in their review that to fully conclude on the effectsof adding prebiotics in fish diets, more research efforts are needed inorder to provide the aquaculture industry, the scientific community,the regulatory bodies and the general public with the necessaryinformation and tools.

4.1. Mannanoligisaccharides (MOS)

MOS are glucomannoprotein-complexes derived from the cell wallof yeast (S. cerevisiae) (Sohn et al., 2000). Its use in terrestrial animalsis well documented (for a review see Benites et al., 2008; Klebaniuk etal., 2008; Yang et al., 2009) but the use of MOS has recently beenintroduced in salmonid culture also (Staykov et al., 2007; Grisdale-Helland et al., 2008; Rodriguez-Estrada et al., 2009; Yilmaz et al.,2007; Dimitroglou et al., 2009).

Grisdale-Helland et al. (2008) evaluated the effect of MOSsupplementation on on-growing Atlantic salmon. Dietary MOS wassupplemented at 1% to salmon for a period of 16 weeks. The resultsshowed that apparent energy digestibility of the MOS supplementeddiet was increased compared with the control treatment. Analysis ofbody composition revealed that gross energy content was increasedwith the addition of MOS but crude protein was reduced. Further-more, whole blood neutrophil oxidative radical production and serumlysozyme activity were reduced in fish fed the MOS supplementeddiet.

In the rainbow trout study by Staykov et al. (2007), growthperformance and immune parameters of fish reared either in freshwater net cages or fresh water raceways were investigated. Comparedto the control fish, 0.2% dietary MOS supplementation increased finalbody weight, reduced FCR and mortalities in both net cage andraceway reared trout. MOS supplementation induced significantimprovements of immune parameters compared to the control

groups. However, variations in the benefits were observed betweennet cage reared trout and raceway reared trout. Serum antibody titre,bactericidal activity and lysozyme activity were significantly in-creased in the net cage reared trout; in the raceway reared trout, MOSsupplementation significantly increased serum lysozyme activity,classical and alternative pathway complement activity. In anotherrainbow trout study, the effect of dietaryMOS (1.5%, 3.0% and 4.5%) ongrowth performance and body composition was investigated byYilmaz et al. (2007). The authors reported increased growthperformance when the fish were fed the lowest (1.5%) MOS level.Conversely, carcass protein content increased with increasing MOSsupplementation. Histological analysis of the proximal intestineshowed that fish fed 1.5% and 3.0% MOS displayed longer villi thanboth the control group and the 4.5% MOS group. However, FCR, PERand hepatosomatic index (HSI) remained unaffected. In an in vivostudy on rainbow trout fingerlings fed 0.4% MOS for 12 weeks,Rodriguez-Estrada et al. (2009) reported that inclusion of MOS to thediet, stimulated growth, haemolytic- and phagocytic activity, mucosaweight and improved survival when the fish were challenged with V.(L.) anguillarum.

The results from a recent studywith juvenile (initial weight∼38 g)and sub-adult (initial weight ∼112 g) rainbow trout reared undercommercial farming conditions demonstrated that dietary MOS had aclear effect on both the intestinal microbiota and histology (Dimi-troglou et al., 2009). Supplementation of 0.2% MOS to the dietsignificantly reduced the aerobic culturable bacterial load within theGI tract. Additionally, changes in the relative abundance of themicrobiota were observed. In the juvenile fish group, MOS fed troutdisplayed a significant reduction of both the relative and absoluteabundance of Micrococcus spp., Aeromonas/Vibrio spp. and a group ofunidentified Gram-positive rods. Coinciding with these changes, asignificant increase of the relative abundance of enterococci wasobserved. Compared to the control group, theMOS fed sub-adult troutdisplayed a significant reduction of Micrococcus spp. and Enterobac-teriaceae and an increase of Pseudomonas spp. Culture-independentanalysis using PCR-DGGE showed that dietary MOS supplementationresulted in reduced bacterial species richness in both juvenile andsub-adult groups compared to the respective controls. Non-metricmultidimensional scaling (nMDS) analysis also demonstrated a clear

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MOS induced effect on the microbiota; dietary MOS caused a distinctspatial shift of bacterial communities away from the control groups.Histological evaluation using light microscopy revealed that the sub-adult MOS group displayed significantly increased intestinal absorp-tive surface in both the proximal and distal intestinal regions.Furthermore, electron microscopy revealed increased microvillidensity in the distal intestinal region and increased microvilli lengthin both intestinal regions in the sub-adult MOS fed trout.

4.2. Inulin

Inulin is a term applied to a heterogeneous blend of fructosepolymers differing from FOS (or oligofructose), a subgroup of Inulin,by the degree of polymerisation (DP): inulin DPN10 and FOS DP≤10(Niness, 1999). Inulin is found naturally in a variety of plant foodssuch as bananas, barley, chicory, garlic, Jerusalem artichoke, leeks,onions and wheat (Roberfroid, 1993; van Loo et al., 1995) and its usein endothermic animals is well documented in several comprehensivereviews (Havenaar et al., 1999; Gibson et al., 2004; Possemiers et al.,2009; Kelly, 2009; Wang, 2009).

The first study using inulin in salmonids was conducted by Olsenet al. (2001), who fed high level (15%) dietary inulin to on-growingArtic charr. After 4 weeks of feeding, histological analysis revealedthat inulin caused destructive effects in the lay-out and structure ofmicrovilli in the distal intestine. Additionally, lamellar bodiesdominated the enterocytes interior and the presence of vacuolesalso increased. Pyloric caeca were also examined and the effect ofinulin was restricted only in the lysosomal bodies which wereincreased from 0.5% to 2.2% in the inulin fed fish.

Some information is available about inulin fermentation by lacticacid bacteria isolated from the fish gut, notably, C. maltaromaticum,Carnobacterium mobile and Carnobacterium spp. (Ringø, 2004). Basedon this finding, a subsequent investigation of the effect of 15% dietaryinulin on the culturable aerobic and facultative aerobic autochthonousmicrobiota of the distal intestine of Arctic charr was conducted using16S rRNA identification, assessment of isolates ability to utilise inulinand electron microscopy (Ringø et al., 2006). Substituting dietarydextrin with inulin reduced the bacterial population and also alteredmicrobial composition. The general findings from this study were thatthe distal gut microbiota of control fish fed (15% dextrin) containedPseudomonas spp., Psychrobacter glacincola, C. divergens. Micrococcusspp., Staphylococcus spp. and Streptococcus spp. while the gutmicrobiota of fish fed 15% inulin comprised of Bacillus spp., C.maltaromaticum, Staphylococcus spp. and Streptococcus spp. However,the ability to ferment inulin was not only restricted to carnobacteriaas some strains of both Staphylococcus spp. and Streptococcus spp.isolated from the control group also had this ability. Although changesin themicrobiota were observed, no clear conclusion can be given thatinulin stimulates colonisation of inulin utilising bacteria in the gut ofArctic charr based on the results of Ringø et al. (2006). Electronmicroscopical analysis of distal intestine confirmed the results oftraditional culture-based microbial analysis as fewer bacterial cellswere observed between microvilli and associated with the surfaces ofenterocytes of fish fed inulin compared to that for the dextrin fed fish.

Refstie et al. (2006) conducted a study to investigate the effect ofdietary inulin (7.5%), with and without oxytetracycline addition(0.3%), on Atlantic salmon for a 3 week period. At the end of the trialblood plasma analysis revealed that the concentration of free fattyacids, glucose, cholesterol and triacylglycerides were not affected bythe dietary inulin or the oxytetracyline supplementation. Fish fedinulin displayed a higher relative gut weight (relative to total bodyweight) but relative liver and stomach weights were unaffected.Histology of the distal intestine revealed that neither inulin noroxytetracycline induced any morphological changes. Additionally,analysis of GI tract for trypsin and amylase activity and brush bordermembrane alkaline phosphatase and leucine aminopeptidase activity

revealed that dietary inulin significantly affected the relative trypsinactivity in the distal intestine, the total amylase activity in theproximal and the distal intestine and the leucine aminopeptidaseactivity of the distal intestine (expressed relative to protein).Apparent amino acid absorption was not affected by inulinsupplementation.

A more recent study with a similar experimental design, dietarysupplements and trial duration to that of Refstie et al. (2006) wasconducted by Bakke-McKellep et al. (2007). The results revealed thatinulin, with or without oxytetracycline addition did not affect finalbodyweight or body length.With the exception of the distal intestinalsomatic index no other gastric organosomatic indices were affected.

Histological evaluation of the proximal and the distal intestinerevealed that neither inulin or oxytetracycline influenced theintestinal morphology. However, the muscle layer of the proximalintestine of inulin fed fish showedmoderate leucocytic cell infiltration(in 6 out of 12 fish) compared with the control fish. Immunohisto-chemical measurements of proliferating cell nuclear antigen demon-strated that inulin supplementation did not affect the positiveproliferative compartment length. However, inulin with oxytetracy-cline addition reduced the proliferative compartment length. Addi-tionally, microbial enumeration of the gut microbiota revealed thatinulin reduced the allochthonous viable bacteria in digesta of both theproximal and distal intestine but autochthonous population levelswere unaffected.

The contrasting findings in gut morphology in the study withArctic charr (Olsen et al., 2001) compared to that reported in Atlanticsalmon (Bakke-McKellep et al., 2007) may be due to dietary levels ofinulin, 15% for Arctic charr vs. 7.5% for Atlantic salmon, or differentanalytic methods; transmission electron microscopy (TEM) vs. lightmicroscopy (LM). LM used by Bakke-McKellep et al. (2007) does notallow sufficient resolution to evaluate changes in the organization ofmicrovilli and the presence of intracellular lamellar bodies in distalintestinal enterocytes as observed by TEM (Olsen et al., 2001). Basedon the results from these two studies we highly recommend that bothLM and TEM are included in future studies.

4.3. Other prebiotics

The commercial product GroBiotic®-A is a mixture of partiallyautolyzed brewers yeast, dairy ingredient components and driedfermentation products (Li and Gatlin 2005). An overview of thestudies carried out using GroBiotic®-A in aquaculture is presented byRingø et al. (2010b), but to our knowledge only one study has beenconducted on salmonids. Sealey et al. (2007) described the effect ofdietary partially autolysed yeast and GroBiotic®-A on rainbow trout.Fingerlings were fed the experimental diets containing 2% prebioticfor 9 weeks. The results showed that growth performance, immuneresponses and TNF-α mRNA expression level remained unaffectedthroughout the experimental period. On the contrary, whole bodyenergy content and survival from infectious hematopoietic necrosisvirus (IHNV) challenge was significantly increased in fish fed eitherpartially autolysed yeast or GroBiotic®-A.

To our knowledge only one study has used FOS on salmonids(Grisdale-Helland et al., 2008). In this study the authors evaluated theeffect of 1% dietary FOS supplementation on on-growing Atlantic salmonafter feeding for 16 weeks. The results demonstrated that FOS fed salmondisplayed some improvement of feed efficiency ratio (FER) compared tothe control group. However, final body weight, apparent nutrientdigestibility and carcass proximate analysis remained unaffected.Additionally, whole blood neutrophil oxidative radical production andserum lysozyme activity remained unaffected. In this study, Grisdale-Helland et al. (2008) also included a treatment of dietary galactooligo-saccharides (GOS) which, to our knowledge, is the only GOS studyconductedwith salmonids (Grisdale-Hellandet al., 2008).Using the sameexperimental protocols, on-growing Atlantic salmon fed 1% dietary

14 D.L. Merrifield et al. / Aquaculture 302 (2010) 1–18

prebiotic for 16 weeks, Grisdale-Helland and colleagues observed thatGOSwasunable to affect growthparameters relative to the control group.Apparent nutrient digestibility and body composition also remainedunaffected. However, body gross energy content of GOS fed fish wasreduced.Whole bloodneutrophil oxidative radical production and serumlysozyme activity remained unaffected with GOS incorporation in thediet.

Readers with special interest in the different aspects on the use ofprebiotics (MOS, FOS, inulin, short-chainFOS,GOS, xylooligosaccharides,arabinoxylooligosaccharides isomaltooligosaccharides) in other fishspecies are referred to the recent review of Ringø et al. (2010b) andthe results of El-Dakar et al. (2007), Burr et al. (2008, 2009) andLochmann et al. (2009).

4.4. Future prebiotic work

Three of the gold standards to be included in future prebiotic studiesare the effect on immune responses, gut microbiota and challengestudies. Very few studies have assessed the effects of prebiotics on theimmune response of salmonid fish (Sealey et al., 2007; Staykov et al.,2007; Rodriguez-Estrada et al., 2009). Based on this factwe suggest that afull understanding of the uses of prebioticswill only be obtained bymoreintensive studies investigating the effects of prebiotics, in modulatingboth rapid-acting innate responses and slower pathogen-specificadaptive immune responses, in the context of both soluble and cellulareffectors relevant to the gut mucosa. This will only adequately beaddressed in comparative studies of healthy and pathogen challengedfish models. In the 70s, 80s and 90s, the majority of investigations of theintestinal microbial communities of fish focused on culturable aerobicpopulations (for review see Cahill, 1990; Ringø et al., 1995; Ringø andBirkbeck, 1999). These investigations can be useful for determining thedominant aerobic and facultative anaerobic bacteria, but are notappropriate for isolating strict anaerobic bacteria. Even when severalselective growth media are used culturable studies still do not present acorrect overview of the gut microbiota. Therefore we recommend thatmolecular methods such as DGGE and FISH are used in future prebioticstudies. To our knowledge, very few studies have used DGGE to evaluatethe effect of prebiotics on gutmicrobial community infish and shrimp (Liet al., 2007; Dimitroglou et al., 2009, 2010; Burr et al., 2009; Zhou et al.,2009). Furthermore, as only one investigation (Rodriguez-Estrada et al.,2009) has included a challenge study in salmonids this topic should alsobe given high priority in future studies.

5. Synbiotics

Synbiotics, the combined application of probiotics and prebiotics,is based on the principle of providing a probiont with a competitiveadvantage (a fermentable energy source) over competing endoge-nous populations; Thus, effectively improving the survival andimplantation of the live microbial dietary supplement in thegastrointestinal tract of the host (Gibson and Roberfroid, 1995). Tothe authors knowledge only one synbiotic study has been conductedin salmonids (Rodriguez-Estrada et al., 2009). In this study theindividual application of the dietary E. faecalis and MOS provided awide range of benefits with regards to immune response and survivalin a challenge study with V. (L.) anguillarum. However, it was notedthat synbiotic feeding (E. faecalis+MOS) yielded significantly betterresults than either individual probiotic or prebiotic application.Compared to the control, probiotic feeding failed to influence growthperformance but MOS however significantly elevated weight gain,SGR and FCR. Interestingly, these parameters were further elevated infish fed the synbiotic diet. In order to clarify similar effects on othersalmonids we recommend that further synbiotic studies should beconducted.

6. Concluding remarks and future perspectives

Current research provides a foundation but applications withinthese studies are often impractical at industrial level farming that it isdifficult to plan feeding strategies for commercial level applications.Future efforts must focus on implementing more practical applica-tions as well as scientific studies designed to understand themechanisms that underpin and mediate the observed host benefits.In this context, growth performance parameters and body composi-tion analysis should be incorporated into future trials. Additionally,digestibility and cost–benefit analysis should be considered; this maybe of particular relevance if selected probiont candidates display keydigestive enzymatic attributes with regards to selected dietarycomponents (plant proteins, chitin etc) which can then be supple-mented to diets containing high levels of host non-digestible material.Although many tend to think of biotic applications in terms of healthand disease benefits we must not overlook the benefits in terms ofaiding host digestive function.

Considering the gaps in the current knowledge, future investigationsof biotic applications for salmonids should expand to more studies onAtlantic salmon and other important salmonids (e.g. Arctic charr, Chinooksalmon Oncorhynchus tshawytscha, coho salmon Oncorhynchus kisutch,brown trout and sea trout S. trutta morpha trutta) as present data isheavily based on rainbow trout. Likewise, research at the larval stage, acritical time in the life cycle, should also be a focal point of concertedinterest as most salmonid probiotic studies are based on juveniles.

The application of probiotics has been demonstrated to improvetolerance of other fish species to environmental stressors, particularly atthe larval stage (Lara-Flores et al., 2003; Taoka et al., 2006b; Rollo et al.,2006; Raj et al., 2008; Gomes et al., 2009). However, to the authorsknowledge there is currently no literature available regarding the effect ofbiotics on salmonids during stress (stress associated with bacterialchallenge studies notwithstanding). It is at these criticalwindows that therole of biotic dietary supplements may offer an effective barrier againstpathogenic invasion and subsequent mortalities; future studies arerequired in order to see if such benefits can be achieved with salmonids.

Acknowledgements

The authors, as well as their respective institutions, would like todedicate this article to their dear departed colleague and friend, BrunoRochet (who died on 5 November 2009 at the age of 55 years). Brunowas the business development director for Lallemand AnimalNutrition, having joined Lallemand in 1998, after numerous years ofworking on the use of probiotics in Animal Nutrition. He was thefounder and first president of the European Probiotic Associationestablished in 1999. As one of the pioneers in the field, the recentEuropean demarche and pursuit of the authorisation and use ofprobiotics in aquaculture could be attributed to his vision andforesight. In the last five years, he had pursued his passion onaquaculture with determined dedication, initiating various researchand developmental projects in the area and, in particular, the use ofprobiotics in aquaculture.

We will all miss this very unique, passionate and dedicatedgentleman who has greatly contributed to the use of probiotics inaquaculture and of course, the development of the Europeanprobiotics business in general.

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