2010 - Current Status Probiotik

Aquaculture 302 (2010) 118
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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Review
The current status and future focus of probiotic and prebiotic applications for salmonids
Daniel L. Merrield a,, Arkadios Dimitroglou a, Andrew Foey b, Simon J. Davies a, Remi T.M. Baker a, Jarl Bgwald c, Mathieu Castex d, Einar Ring c
a
Aquaculture and Fish Nutrition Research Group, School of Marine Science and Engineering, Marine Institute, University of Plymouth, UK School of Biomedical and Biological Sciences, University of Plymouth, Plymouth, UK Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, University of Troms, Norway d Lallemand Animal Nutrition, Blagnac, France
b c
a r t i c l e
i n f o
a b s t r a c t
Salmonids are an important contributor to sh production in many countries. Concerted research efforts have concentrated on optimising production with eco-friendly alternatives to the therapeutic use of antimicrobials. Probiotics and prebiotics offer potential alternatives by providing benets to the host primarily via the direct or indirect modulation of the gut microbiota. Suggested modes of action resulting from 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, inhibition of virulence gene expression, enhancing the immune response, improved gastric morphology and aiding digestive function. The application of probiotics and prebiotics may therefore result in elevated health status, improved disease resistance, growth performance, body composition, reduced malformations and improved gut morphology and microbial balance. Current research demonstrates successful proof of these concepts and a foundation for applications in salmonid aquaculture. However, application strategies applied in current studies are varied and often impractical at industrial level farming; thus, it is difcult to plan an effective feeding strategy for commercial level applications. Future studies should focus on providing practical industrial scale applications. Additionally, from a scientic perspective we must have a better understanding of the mucosalbacterial interactions which mediate the host benets in order to achieve optimal utilisation. 2010 Elsevier B.V. All rights reserved.
Article history: Received 22 November 2009 Received in revised form 4 February 2010 Accepted 5 February 2010 Keywords: Disease Salmon Trout Synbiotic Microbiota Health
Contents 1. 2. 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Endogenous microbiota, mucosal tolerance and development . . . . Probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Probiotic selection criteria . . . . . . . . . . . . . . . . . 3.2. Mode of action and reported probiotic benets in salmonids . 3.3. Immune response . . . . . . . . . . . . . . . . . . . . . 3.4. Disease resistance . . . . . . . . . . . . . . . . . . . . . 3.4.1. Viral diseases . . . . . . . . . . . . . . . . . . . 3.4.2. Bacterial diseases . . . . . . . . . . . . . . . . . 3.4.3. Disease challenge methods . . . . . . . . . . . . 3.5. Effect of probiotics on gut microbiota . . . . . . . . . . . . 3.6. Gastrointestinal morphology . . . . . . . . . . . . . . . . 3.7. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Feeding strategies . . . . . . . . . . . . . . . . . . . . . 3.8.1. Choosing a probiont. . . . . . . . . . . . . . . . 3.8.2. Supplementation form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 3 3 3 4 4 4 5 6 6 8 9 9 9 9
Corresponding author. Tel.: + 44 1752 584 877; fax: +44 1752 584950. E-mail address: daniel.merrield@plymouth.ac.uk (D.L. Merrield). 0044-8486/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.02.007
D.L. Merrield et al. / Aquaculture 302 (2010) 118
3.8.3. Vector of administration . . 3.8.4. Dosage level . . . . . . . 3.8.5. Supplementation duration . 3.9. Issues with industrial level scale-up . 3.10. Future probiotic work . . . . . . . 4. Prebiotics . . . . . . . . . . . . . . . . 4.1. Mannanoligisaccharides (MOS) . . . 4.2. Inulin . . . . . . . . . . . . . . . 4.3. Other prebiotics . . . . . . . . . . 4.4. Future prebiotic work . . . . . . . 5. Synbiotics . . . . . . . . . . . . . . . . 6. Concluding remarks and future perspectives Acknowledgements . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .
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1. Introduction Although there is now global recognition that aquaculture production is expanding to a wide diversity of cultured nsh, salmonids remain an important contributor to sh production in many countries. Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss W.) are reared principally in Norway, Scotland and Chile with some output arising from Canada, the USA, regions of Europe and to a lesser extent Australia and New Zealand. Total global production of salmonids was reported to exceed 2.2 million mt in 2007 (FAO, 2009). These species attract high market prices and are a stable source of high quality sh due to consumer acceptance for quality seafood. Salmon in particular are noteworthy for their characteristic pink-reddish esh pigmentation (Davies, 2008) and versatility with respect to processing into a wide range of food products. Rainbow trout is also a popular salmonid sh species and has received great attention in terms of importance to aquaculture and remain a core of inland sh production in many countries throughout the world. It should also be recognised that native species of brown trout (Salmo trutta) are important especially in terms of their contribution to recreational sheries and angling. Additionally, developments in rearing techniques for Arctic charr (Salvelinus alpinus) have increased interest in commercial farming and consequently global production has risen to over 2000 mt in 2007 (Jobling et al., 1993; FAO, 2009). Collectively, there has been an abundance of scientic literature underpinning the genetics, nutrition, health and disease issues concerning the development of the salmonid aquaculture industry. Given the importance of nutrition in maintaining the health of sh, with respect to nutritional involvement on immuno-competence and disease resistance, as well as its role in stress mediation, there is a growing trend towards exploring dietary components of a nonnutritional nature to provide various functional attributes. This has been compounded by the constraints of employing antibiotics in the aquaculture industry, as reected by the EU moratorium on the banning of antibiotic growth promoters in animal feeds, including sh (Regulation, EC No, 1831). There have been numerous investigations on salmon and trout to evaluate the feasibility of supplementing diets with a range of potentially probiotic bacteria. Several general reviews have been published over the past decade summarising the latest available literature (Ring and Gatesoupe, 1998; Gatesoupe, 1999; Ring and Birkbeck, 1999; Vershuere et al., 2000; Irianto and Austin, 2002a; Ring, 2004; Burr et al., 2005; Balczar et al., 2006a;Gram and Ring, 2005; Ring et al., 2005; Gatesoupe, 2007; Kesarcodi-Watson et al., 2008; Wang et al., 2008a). Furthermore, specic reviews have focused on larvae (Gomez-Gil et al., 2000; Vine et al., 2006; Tinh et al., 2008),
shrimp (Farzanfar, 2006; Ninawe and Selvin, 2009), shellsh (Balczar et al., 2006b) along with reviews specic 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 forward to summarise the effects of probiotics on salmonids. Additionally, with the growing interest and assessment of prebiotic applications for sh, it is pertinent to review the present ndings with regards to salmonid sh. The aim of this review is to evaluate the literature currently available regarding the use of probiotics and prebiotics (collectively referred to as biotics hereafter) on salmonids. Specic emphasis is placed on highlighting application strategies on a practical basis and potential future research. In order to discuss these issues we must rst examine the complex microbehost interactions within the gut, which ultimately inuence the health and development of the host. 2. Endogenous microbiota, mucosal tolerance and development The immune system of teleost sh appears to be an efcient means by which the host protects itself upon pathogenic challenge. But not all microbes represent a pathogenic threat; resident commensal microbes help maintain efcient functioning of the gut by supporting gut mucosal barrier function: mounting efcient immune responses to pathogens that break through barrier defences or maintaining tolerance (i.e. immune non-responsiveness) to luminal contents which allow for nutrient absorption. The inter-relationship between gut mucosal epithelial cells, mucus, anti-microbial products, commensal organisms resident in the gut and immune cells in the mucosa/sub-mucosa are vital for the health and well-being of the sh. These interactions prime regulatory mechanisms which result in mucosal tolerance. From our understanding of mammalian systems, this induction of tolerance may result from selective antigen tasting of luminal contents by specialised antigen presenting cells (either epithelial cells or dendritic cells) which prime regulatory T cells, which, in turn, suppress effector T cell responses. These suppressive responses are broken upon sensing of danger signals, presented upon pathogen invasion of mucosa/sub-mucosa (Mason et al., 2008). No such regulatory T cells have been characterised as yet, in teleost sh. The characterisation of the regulatory cytokines (anti-inammatory) IL-10 and TGF, however, is suggestive that such mechanisms may, in fact, exist. Endogenous commensal microbiota play an important role in tolerance induction versus immune activation decisions. Although a great deal of further research into these complex interactions is required, Gmez and Balczar (2008) recently provided a review of our present level of understanding on the interactions between gut microbiota and innate immune system of sh. Our knowledge of the importance of the endogenous microbiota on mucosal development and maturation of the immune system is
expanding, in part, due to recent studies using zebrash (Danio rerio). The zebrash has become an integral model species responsible for much of our understanding of the genetics underpinning sh development and functionality (Dahm and Geisler, 2006). Furthermore, several studies have demonstrated the importance of the complex microbehost interactions on gastric development (Rawls et al., 2004, 2007; Bates et al., 2006). For example, zebrash gene expression has been demonstrated to be inuenced by microbial colonisation, assessed by comparing conventionally reared and germ-free zebrash larvae (Rawls et al., 2004). Rawls et al. (2004) observed that microbiota stimulated intestinal epithelial proliferation and inuenced enterocyte morphology. Furthermore, expression of serum amyloid A1 and complement component 3 genes were elevated in conventionally reared larvae compared to germ-free larvae. As certain genes were always expressed, irrelevant of the bacterial coloniser, and the expression of other genes was bacteria-specic, the authors concluded that a subset of zebrash genes were responsive to factors present only in a subset of bacterial groups. Additionally, Bates et al. (2006) reported that the gut epithelial mucosa failed to differentiate fully in germ-free zebrash larvae. A lack of brush border alkaline phosphatase activity, immature patterns of glycan expression and a distinct reduction of goblet and enteroendocrine cells was observed in germ-free larvae compared to conventionally reared larvae. However, reintroduction of microbiota reversed these phenotypic changes. Therefore, manipulation of endogenous microbial populations by probiotic or prebiotic supplementations could have appreciable effects on immunoregulatory mechanisms behind gut mucosal tolerance. 3. Probiotics The word probiotic is constructed from the Latin word pro (for) and the Greek word bios (life) (Zivkovic, 1999). The denition of a probiotic differs greatly depending on the source, but the rst generally accepted denition was proposed by Fuller (1989) as a live microbial feed supplement which benecially affects the host animal by improving its microbial balance. Given the nature of sh farming and the fact that water harbours microbial communities it is accepted that we must have a distinctive denition for aquatic animals as opposed to that proposed by Fuller (1989) for terrestrial animals. The evolution of the denition for aquaculture throughout the 1990s is discussed by Gomez-Gil et al. (2000). During this period the denition was rened and new terminology for microbial applications in aquaculture were proposed. Microbes that are antagonistic to pathogens, but are not found to be present, 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 by the breakdown of waste or pollutants have been termed bioaugmentation or bioremediation (Moriarty, 1997, 1998; Gatesoupe, 1999). Thereafter, many authors proposed denitions for probiotics in aquaculture that were inclusive of administration via rearing water but tended to restrict applications to microbes that were associated with health promoting properties (Spanggaard et al., 2001; Irianto and Austin 2002a). Contrary to these denitions, some denitions do not focus only on health benets (Moriarty, 1998; Gram et al., 1999; Vershuere et al., 2000; Farzanfar, 2006). Whilst many people now refer to all of these microbial applications as probiotic treatments (sensu lato), it is important to distinguish the differences between them. If we were to merge all of the proposed denitions it would appear that a probiotic application for aquaculture is a live, dead or component of a microbial cell that when administered via the feed or to the rearing water benets the host by improving either disease resistance, health status, growth performance, feed utilisation, stress response or general vigour, which is achieved at least in part via improving the hosts microbial balance or the microbial balance of the ambient environment. If we are to restrict the benets to the host to improvements of health, then we must devise new terminology for microbes that
improve growth performance or feed utilisation, microbes that modulate the gut microbiota without stimulating the immune response (which may or may not provide a benet by preventing ubiquitous potential pathogens from colonising the digestive tract), microbes that improve gastric morphology or function and microorganisms that prevent malformation or improve the quality of the nal product. As there are no proposed denitions for such organisms, and for the sake of the current review we will suppose that a probiotic is any microbial cell provided via the diet or rearing water that benets the host sh, sh farmer or sh consumer, which is achieved, in part at least, by improving the microbial balance of the sh. In this context we regard direct benets to the host as immuno-stimulation, improved disease resistance, reduced stress response, improved GI morphology etc and benets to the sh farmer or consumer as improved sh appetite, growth performance, feed utilisation, improvements of carcass quality, esh quality and reduced malformations. 3.1. Probiotic selection criteria Previous reviews have proposed favourable characteristics for the selection of potential probionts for applications with sh species (Spanggaard et al., 2001; Balczar et al., 2006a; Vine et al., 2006; Farzanfar, 2006; Gmez and Balczar, 2008). Following on from these papers we propose an extended list of criteria for potential probionts, some of which are essential (E) and some considered as merely favourable (F). The more of these characteristics that are fullled by a candidate probiotic species, the more appropriate that species shall be considered and thus more likely to be an effective sh probiont: It must not be pathogenic, not only with regards to the host species but also with regards to aquatic animals in general and human consumers (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 intestinal mucus (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 lag period, a short doubling time and/or growth at host rearing temperatures) (F) should exhibit antagonistic properties towards one or more key pathogens: in the case of salmonids one might focus on Aeromonas salmonicida, 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 the diet or cellulase if the diet is rich in plant ingredients) or produce vitamins. (F) should be indigenous to the host or the rearing environment (F) should remain viable under normal storage conditions and robust enough to survive industrial processes (F) As it is unlikely to nd a candidate that will full all of these characteristics we should begin to further explore the possibilities of simultaneously using several probiotics or the use of probiotics with prebiotics (termed synbiotics) (Patterson and Burkholder, 2003). Through the combined application of multiple favourable probiotic candidates it may be possible to produce greater benets (and satisfy more of the previously suggested characteristics) than the application of individual probionts. 3.2. Mode of action and reported probiotic benets in salmonids The specic mode of action resulting in the observed host benets is often difcult to elucidate conclusively, due to the wide range of possible modes of action and the complicated synergistic multi-factorial
relationships between them. Thus, much of our understanding of the mechanisms behind probiotic effects stem from human studies. However, suggested probiotic modes of action in sh include: production of inhibitory compounds, competition for chemicals or available energy, competition for adhesion sites, inhibition of virulence gene expression or disruption of quorum sensing, improvement of water quality, enhancement of the immune response, source of macro and/or micronutrients and enzymatic contribution to digestion (Sugita et al., 1996, 1997; 1998; Vershuere et al., 2000; Olafsen, 2001; Vine, 2004; Gatesoupe 2008; Gmez and Balczar, 2008; Ring, 2008; Tinh et al., 2008). An extended discussion of the mechanisms of action of probiotic applications in sh is presented by Tinh et al. (2008). Despite not always being fully aware of the precise mode of action it is clear that probiotic applications with salmonids have resulted in elevated health status, improved disease resistance, growth performance, feed utilisation, carcass composition, gastric morphology, reduced malformations, GI colonisation and subsequently microbial modulation. For a summary of probiotic applications in salmonids refer to Table 1. 3.3. Immune response Despite the large number of probiotic studies which have assessed immunological and haematological parameters (Panigrahi et al., 2004, 2005, 2007; Merrield et al., 2010a,b,c; Kim and Austin, 2006a,b; Brunt et al., 2007, 2008; Irianto and Austin, 2002b; Balczar et al., 2006a,b; Nikoskelainen et al., 2003; Raida et al., 2003; Arijo et al., 2008; Pieters et al., 2008) the immuno-modulatory effects of probiotics in sh systems are, at present, poorly understood. Probiotic strains of Carnobacterium have been demonstrated to augment innate immune responses in rainbow trout, upon challenge with sh pathogens 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 and Austin, 2006b), investigated the effects of the same probiotic strains on cytokine expression (IL-1, IL-8, TNF and TGF) by head kidney (HK) leukocytes and gut cells. None of the strains induced cytokine expression in gut cells but augmented the expression of IL-1 and TNF in HK leukocytes suggestive of a strengthening of innate immunity in rainbow trout (Kim and Austin, 2006b). In a separate study 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 enhanced superoxide anion production and serum alternative complement activity. In addition, the expressions of IL-1 , TNF and TGF were also upregulated in the spleen and HK. Such results are again suggestive of augmentation of innate immunity and possibly regulatory mechanisms behind mucosal tolerance. The effects on the gut mucosal tissue, however, were not studied. Balczar et al. (2007b) administered dietary strains of Lactococcus lactis, Lactobacillus sakei and Leuconostoc mesenteroides to rainbow trout for 2 weeks at 106 CFU g 1. Compared to the control group, HK leukocyte phagocytic activity and serum alternative complement activity were greater in probiotic fed sh groups. In the case of L. sakei, superoxide anion production was also enhanced; however, no effect on lysozyme activity was detected. The activity of serum lysozyme and alternative complement are typical parameters assessed when investigating the effect of probiotics on sh innate immune response (Panigrahi et al., 2004, 2005; Kim and Austin, 2006a,b; Balczar et al., 2006a,b; Newaj-Fyzul et al., 2007; Merrield et al., 2010a,b). Probiotics (Bacillus spp. and LAB) often induce signicant elevations of trout serum lysozyme activity (NewajFyzul et al., 2007; Balczar et al., 2007a; Merrield et al., 2010a); however, the current ndings are somewhat unclear as several studies have demonstrated variable effects of probiotics on serum lysozyme activity (Panigrahi et al., 2004, 2005; Balczar et al., 2007a, b; Merrield et al., 2010a,b).
With regards to probiotic effects on the adaptive immune system, Arijo et al. (2008) demonstrated that the administration of live probiotic strains resulted in the expression of cross-reactive antibodies which were specic for outer membrane proteins and extracellular products of bacterial pathogens, conferring a protective effect upon challenge with Vibrio harveyi. In addition, the same group also demonstrated that dietary probiotics (Aeromonas sobria strain GC2) can protect rainbow trout against skin infections caused by the Aeromonas bestiarum (n rot) and the eukaryotic pathogen Ichthyophthirius multiliis (white spot) (Pieters et al., 2008). This protection arose as a consequence of augmentation of the phagocytic response whereas a second protective probiotic strain enhanced respiratory burst activity (Pieters et al., 2008). A more comprehensive overview of probiotics and disease resistance in salmonids is provided in the next section. From these studies, it would appear that probiotics enhance innate immunity and that the innate pathway response was dependent on the probiotic strain used. In addition to direct effects, indirect effects of probiotics may confer health benets. Fermentation products such as the short-chain fatty acid, butyrate, both modulate barrier function and regulate inammatory processes, by decreasing epithelial permeability through up-regulation of tight junction proteins and suppression of pro-inammatory cytokines by induction of expression of antiinammatory, regulatory cytokines, respectively (van Nuenen et al., 2005). These short-chain fatty acids (SCFAs) are effectively acting as adopted regulators of both innate and adaptive immune mechanisms. The current literature is indicative of probiotic dietary supplements effectively boosting innate immune defences and adaptive immune mechanisms of sh. So far, limited responses observing upregulation of TGF and IL-10 expression are only suggestive of a probiotic modulatory effect on maintenance of mucosal tolerance, crucial in tolerating the useful and mounting responses to the pathogenic. Thus, it would be fair to conclude that, similar to many commercially available probiotic preparations in use for humans, the best level of protection towards a broad spectrum of sh pathogens will only be obtained through the use of probiotic dietary preparations consisting of several probiotic strains, each strengthening a specic area of the immune system. 3.4. Disease resistance Both salmon and trout remain susceptible to a number of bacterial and viral diseases (these are summarised in Table 2 and are reviewed in detail by Groff and LaPatra (2000) and Toranzo et al. (2005)) as well as a number of husbandry related stressors such as handling and transportation that are of importance at different stages of growth and production. 3.4.1. Viral diseases The 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 inammation (HSMI), and cardiomyopathy syndrome (CMS). Vaccination is the traditional method for controlling such viral diseases but the level of success is variable. For example, DNA vaccines against IHN and VHS have been developed and demonstrated to give protection against disease (Lorenzen et al., 2000; Kurath et al., 2006). However, despite extensive vaccination of farmed Atlantic salmon against IPN in Norway (with some indications that vaccination may reduce losses) the efcacy of the vaccines in the eld remains disputed (Wetten et al., 2007). Additionally, commercial vaccines against PD have been in development and they have proved to be effective in some eld trials, but the duration of immunity has been questioned (McLoughlin and Graham, 2007). Furthermore, the causes of HSMI and CMS, which have been demonstrated to be infectious, are still under investigation and thus no vaccines are available (Poppe and Seierstad, 2003;
D.L. Merrield et al. / Aquaculture 302 (2010) 118 Table 1 Summary of potential probiotic applications for salmonids. Potential probiont (sh species) Oncorhynchus mykiss La. lactis, Leu. Mesenteroides, L. sakei Leu. mesenteroides, L. plantarum L. rhamnosus L. rhamnosus L. rhamnosus L. rhamnosus L. rhamnosus, B. subtilis, E. faecium E. faecalis B. subtilis B. subtilis + B. licheniformis B. subtilis + B. licheniformis B. subtilis + B. licheniformis, E. faecium B. subtilis + B. licheniformis, E. faecium, P. acidilactici Bacillus spp., A. sobria Bacillus spp., A. sobria P. acidilactici P. acidilactici, S. cerevisiae P. acidilactici, S. cerevisiae C. divergens, C. maltaromaticum C. divergens, C. maltaromaticum C. inhibensa A. sobria A. sobria, Brochothrix thermosphacta A. sobria A. hydrophila, V. uvialis, Carnobacterium spp., C. inhibens, V. alginolyticus and an unidentied Gram-positive coccus A. hydrophila, V. uvialis, Carnobacterium spp., and unidentied Gram-positive coccus Ps. uorescens, Pseudomonas strains, Carnobacterium strains Ps. uorescens S. cerevisiae,D. hansenii,R. glutinis S. cerevisiae Kocuria SM1 Enterobacter cloacae, Bacillus mojavensis Salmo trutta La. lactis, Leu. Mesenteroides, L. sakei La. lactis, Leu. Mesenteroides, Salmo salar C. inhibensa C. divergens L. delbrueckii C. divergens Ps. uorescens Ps. uorescens V. alginolyticus Parameters investigated DR, IR, GM DR, GM, GP DR, GP GM, IR GM, IR GM, IR IR BC, GP, IR, DR DR, GM, IR DR, IR BC, FU, GM, GP BC, FU, GM, GP, IR GH, GM DR, IR IR BC, FU, GM, GP, IR BC, FU, GM, GP, SM DR, IR IR IR DR, GM DR, IR DR, IR DR, IR DR, IR, GM References Balczar et al. (2007b)a Vendrell et al. (2008)a Nikoskelainen et al. (2001)a Nikoskelainen et al. (2003)a Panigrahi et al. (2004)a Panigrahi et al. (2005)a Panigrahi et al. (2007)a,b Rodriguez-Estrada et al. (2009)a Newaj-Fyzul et al. (2007) Raida et al. (2003)a Bagerhi et al. (2008)a Merrield et al. (2009ab)a Merrield et al. (2010d)a Brunt et al. (2007)a Brunt et al. (2008)a Merrield et al. (2009c)a Aubin et al. (2005ab)a Quentel et al. (2004)a Kim and Austin (2006a)a Kim and Austin (2006b)b Robertson et al. (2000)a Brunt and Austin (2005)a,b Pieters et al. (2008)a Arijo et al. (2008)a,b Irianto and Austin (2002b)a
DR, IR DR DR GM BE, GM DR, IR DR, IR IR, GM DR, IR DR DR, GM, GP GH, DR, GM GH, DR, GM DR DR DR
Irianto and Austin (2003)a Spanggaard et al. (2001)a,b Gram et al. (1999)a,b Andlid et al. (1995)a Wach et al. (2006)a Sharifuzzaman and Austin (2009)a Capkin and Altinok (2009)a Balczar et al. (2007a)a Balczar et al. (2009)a Robertson et al. (2000)a Gildberg et al. (1995)a Salinas et al. (2008)a Ring et al. (2007)b Gram et al. (2001)a,b Smith and Davey (1993)a 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 of probiont colonisation), GP growth performance, IR immunological/haematological response, SM skeletal malformation. a In vivo studies. b In vitro experiments. c Field experiments. 1 Carnobacterium strain K1 has been identied as Carnobacterium inhibens (Jborn et al., 1999).
Kongtorp et al., 2004). An alternative approach may include probiotics but the potential for probiotic applications to improve resistance against viral infections of sh are not presently known. If probiotics can elevate immune status, it is not unreasonable to speculate that this may reduce susceptibility to viral infections. Probiotics, such as L. rhamnosus, have been demonstrated to reduce rotavirus and poliovirus infections in human patients (Marteau et al., 2001; de Vrese et al., 2005). de Vrese et al. (2005) observed that L. rhamnosus increased poliovirus neutralizing antibody titers (NT) and affected the formation of poliovirus-specic IgA and IgG in human patients. Moreover, feed supplementation with a Bacillus megeterium strain has resulted in increased resistance to white spot syndrome virus (WSSV) in shrimp Litopenaeus vannamei (Li et al., 2009). Even if further research is necessary, the authors suggested that immuno-stimulation
is probably one of the important factors for the probiotic bacterium to enhance resistance of shrimp to WSSV. 3.4.2. Bacterial diseases It is generally accepted that in sh, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) GI tract (Ldemel et al., 2001; Ring et al., 2004; Birkbeck and Ring, 2005; Ring et al., 2007a). If the GI tract is involved as an infection route, mucosal adhesion is considered to be a critical early phase in all infections caused by pathogenic bacteria (Knudsen et al., 1999; Namba et al., 2007). When the bacteria are able to colonise the intestinal mucus, they can cross the GI tract lining by transcellular or intracellular routes. Extensive research has begun to demonstrate the efcacy of using probiotics to improve disease resistance and reduce mortalities of
6 Table 2 Important viral and bacterial diseases of salmonid sh. Disease Viral Infectious pancreatic necrosis (IPN) Viral hemorrhagic septicemia (VHS) Infectious hematopoietic necrosis (IHN) Pancreas disease (PD) Infectious salmon anaemia (ISA) Heart and skeletal muscle inammation (HSMI) Cardiomyopathy syndrome (CMS) Bacterial Furunculosis Vibriosis Cold water vibriosis Piscirickettsiosis Enteric redmouth disease (ERM)/yersiniosis
Causative agent IPN virus VHS virus IHNvirus PD virus/salmonid alpha virus (SAV) ISA virus HSMI virus CMS virus? Aeromonas salmonicida Vibrio (Listonella) anguillarum Vibrio salmonicida Piscirickettsia salmonis Yersinia ruckeri
Treatment Vaccine DNA vaccine DNA vaccine Vaccine Vaccine (Faroe Islands) No No Vaccine Vaccine Vaccine No Vaccine
Species S. salar O. mykiss O. mykiss S. salar O. mykiss S. salar S. salar S. salar S. salar O. mykiss S. salar Oncorhynchus S. salar S. salar Oncorhynchus Oncorhynchus Salmo sp. Salvelinus sp. Oncorhynchus S. salar S. salar O. mykiss
Reference Smail et al. (1992) Lorenzen et al. (2000) Lorenzen and LaPatra (1999) McLoughlin and Graham (2007) Mjaaland et al. (1997) Kongtorp et al. (2004) Ferguson et al. (1990) Toranzo et al. (2005) Toranzo et al. (2005) sp. Bruno et al. (1986) Mauel and Miller (2002) sp. sp. Furones et al. (1993)
Bacterial kidney disease Winter ulcer
Renibacterium salmoninarum Moritella viscosa
No Vaccine
sp.
Evenden et al. (1993) Benediktsdottir et al. (2000)
salmonids against bacterial diseases. Among the most important bacterial diseases of salmonids are: furunculosis, vibriosis, cold water vibriosis, piscirickettsiosis, enteric redmouth disease (ERM) [aka yersiniosis], bacterial kidney disease and winter ulcer (Table 2). A summary of probiotic studies assessing disease resistance in salmonids is displayed in Table 3. These studies provide a solid foundation of our knowledge regarding the probiotic potential to reduce aquaculture related diseases and it has been demonstrated that applications of viable, formalised, sonicated cells and cell-free supernatants are potentially effective at reducing mortalities induced by a range of bacterial pathogens (e.g. A. salmonicida, V./L. anguillarum, V. ordalii and La. garvieae). However, we know very little regarding efcacy against other important salmonid bacterial diseases such as V. salmonicida, Piscirickettsia salmonis, R. salmoninarum and Moritella viscosa. Future studies would do well to focus on these pathogens. Although mineral oil-adjuvanted injection vaccines are by far the most efcient, giving rise to protection against diseases, the use of these vaccines often results in adverse side effects including extensive adhesions and pigmentation of the peritoneum (Mutoloki et al., 2006). Therefore, alternative eco-friendly treatments must be considered. Treatments that may have a less signicant environmental impact include the strategic use of immuno-stimulants, probiotics and prebiotics. 3.4.3. Disease challenge methods In 2003, Ring and co-authors put forward the statement the microbiologist must think as the military and use military strategy. If we are going to defeat our enemies, the pathogens, we have to be at the same place and time as them. If probiotic bacteria mostly colonise the pyloric caeca, the probionts will have no effect if the pathogens mostly colonises the mid or hindgut regions and translocate in these regions (Ring et al., 2003). Based on this statement one might ask the question how can we investigate the interactions between the benecial bacteria vs. the pathogens in the GI tract of sh and observe the level of host infection? With this in mind we must consider that the potential of probiotics may be greater than some previous studies appear to suggest. The intraperitoneal (IP) method of disease challenge overrides one of the possible methods of probiotic protection against
pathogens by masking the potential effect of probiotic competitive exclusion within the GI tract. Gastric probiotics may reduce or even prevent gastric infection. IP challenges do not reect the effect of probiotics on resistance to infection; rather they demonstrate the effect of probiotics on infected sh. Therefore, it is recommended that immersion or cohabitation studies are conducted in future challenge experiments in order to truly assess the full potential of candidate probionts. The importance of theses potential antagonistic interactions in the gut are highlighted in a recent review, devoted to LAB vs. pathogens in the digestive tract of sh (Ring et al. 2010a). An additional aspect to consider in disease challenge studies is the feeding duration with the probiotic prior to the challenge. Unfortunately, the effect of feeding duration on probiotic efcacy remains scarcely investigated. Indeed in virtually all previously reported studies (Table 3), probiotics were administrated only for a few weeks prior to the challenge. To our knowledge, only one study in salmonids has assessed the effect of long-term probiotic feeding prior to disease challenge (Quentel et al., 2004). This study, nancially supported by the OFIMER (Ofce National Interprofessionnel des Produits de la Mer et de l'Aquaculture), investigated the efcacy of applying P. acidilactici and Saccharomyces cerevisiae var. boulardii, alone or in combination, on the resistance of rainbow trout after intraperitoneal injection with Y. ruckeri. The rainbow trout used in this experiment came from a preliminary study (Aubin et al., 2005b) where they were fed the probionts from rst feeding to 4 months of age. In the groups of sh fed diet supplemented with probiotic bacteria or yeast, a signicant reduction in accumulative moralities was observed compared with sh fed commercial diet, at all challenge doses, thus demonstrating that S. cerevisiae as well as P. acidilactici were found to signicantly increase the protection of rainbow trout to experimental yersiniosis. Interestingly no production of antibody against Yersinia was detected in any feeding groups, and thus the authors suggested that the protection observed after the challenge may be consecutive of an activation of the innate immune system. 3.5. Effect of probiotics on gut microbiota In recent years, several reviews have suggested that the intestinal microbiota of sh may play a role as a defensive barrier against enteric
Table 3 Probiotic applications for improving disease resistance of salmonids. Challenge method A. sobria Yes Potential probiont Reduced Notes mortalities References
Disease
Causative agent
Species
Streptococcosis S. iniae
O. mykiss IP
Lactococcosis
S. iniae La. garvieae
IP O. mykiss IP
A. sobria, Bacillus spp. A. sobria
Yes Yes
Furunculosis
La. garvieae La. garvieae A. salmonicida A. salmonicida CO Yes Yes Yes Yes No No Yes Yes NA NA Yes Yes/no Yes Yes Yes Yes Dead probiotic cells used Reduced fry and ngerling mortalities
IP CO O. mykiss IP IM, CO, IP
Yes Yes Yes Yes
Live cells, disrupted cells and cell free supernatant effective Brunt and Austin (2005) Formalised cells ineffective Brunt et al. (2007) Live cells, disrupted cells and cell free supernatant effective Brunt and Austin (2005) Formalised cells ineffective Brunt et al. (2007) Vendrell et al. (2008) Brunt et al. (2007) Reduced fry and ngerling mortalities Irianto and Austin (2002b) Irianto and Austin (2003) Balczar et al. (2007b) Nikoskelainen et al. (2001) Robertson et al. (2000) Robertson et al. (2000) Gildberg et al. (1995) Gram et al. (2001) Smith and Davey (1993) Austin et al. (1995) Ring et al. (2007b) Salinas et al. (2008) Balczar et al. (2009) Spanggaard et al. (2001) Gram et al. (1999) Brunt et al. (2007) Rodriguez-Estrada et al. (2009) Reduced mortalities even further when administered in conjunction with dietary MOS
A. salmonicida
A. salmonicida A. salmonicida A. salmonicida A. salmonicida A. salmonicida A. salmonicida A. salmonicida A. salmonicida A. salmonicida A. salmonicida A. salmonicida Pseudomonas spp., Carnobacterium spp. Ps. uorescens A. sobria, Bacillus spp. E. faecalis
A. sobria, Bacillus spp. Leu. mesenteroides, L. plantarum A. sobria, Bacillus spp. A. hydrophila, Vibrio spp., Carnobacterium spp., and an unidentied Gram-positive coccus A. hydrophila, V. uvialis, Carnobacterium spp., and an unidentied Gram-positive coccus La. lactis, Leu. mesenteroides and L. sakei L. rhamnosus C. inhibensa C. inhibensa C. divergens Ps. uorescens Ps. uorescens V. alginolyticus C. divergens L. delbrueckii La. lactis, Leu. mesenteroides Increased mortalities Ps. uorescens added to tank water Ps. uorescens added to tank water Added to tank water Prevent to some extent pathogen-induced gut damage Prevent to some extent pathogen-induced gut damage Water temperature increased progressively from 14 C to 16 C to induce the disease Potential probionts added to tank water Ps. uorescens added to tank water
Vibriosis
V. anguillarum V. anguillarum V. anguillarum V. anguillarum
CO CO CO S. salar CO CO CO IM in vitro in vitro S. trutta Asymptomatic carrier O. mykiss IM IM IP IP
Added to tank water Prevent to some extent pathogen-induced gut damage Added to tank water
ERM
V. ordalii V. harveyi V. anguillarum V. anguillarum V. anguillarum V. ordalii V. ordalii Y. ruckeri Y. ruckeri Y. ruckeri Y. ruckeri Y. ruckeri
IP IP S. salar CO IM in vitro CO IM O. mykiss IP IP CO IM IP
A. sobria, Bacillus spp. A. sobria C. inhibensa V. alginolyticus C. divergens C. inhibensa V. alginolyticus A. sobria, Bacillus spp. B. subtilis + B. licheniformis C. inhibensa Enterobacter cloacae, Bacillus mojavensis P. acidilactici; S. cerevisiae var. boulardii
Yes Yes No Yes NA Yes Yes Yes Yes Yes Yes Yes
Dietary administration of the probiotic strains for 4 months before the challenge Yes Yes Yes Yes
Fin rot White spot B. subtilis
S. salar CO O. mykiss ID O. mykiss IM
C. inhibensa A. sobria, Brochothrix thermosphacta A. sobria, Brochothrix thermosphacta
Brochothrix thermosphacta not effective Viable, formalised, disrupted cells and cell-free supernatant effective
Brunt et al. (2007) Arijo et al. (2008) Robertson et al. (2000) Austin et al. (1995) Ring et al. (2007b) Robertson et al. (2000) Austin et al. (1995) Brunt et al. (2007) Raida et al. (2003) Robertson et al. (2000) Capkin and Altinok (2009) Aubin et al. (2005b), Quentel et al. (2004) Robertson et al. (2000) Pieters et al. (2008) Pieters et al. (2008) Newaj-Fyzul et al. (2007)
Other
Y. ruckeri A. bestiarum Ichthyophthirius multiliis Aeromonas sp.
O. mykiss IP
Bacterial genera abbreviations: A. = Aeromonas, B. = Bacillus, C. = Carnobacterium, L. = Lactobacillus, La. = Lactococcus, Leu. = Leuconostoc, Ps. = Pseudomonas, S. = Streptococcus, V. = Vibrio. Challenge method abbreviations: IP = intraperitoneal injection, ID = intradermal injection, IM = immersion, CO = cohabitation, NA = not applicable. a Carnobacterium strain K1 has been identied as Carnobacterium inhibens (Jborn et al., 1999). 7
infections (Birkbeck and Ring, 2005; Ring et al., 2005; Gmez and Balczar, 2008; Ring and Olsen, 2008; Ring et al., 2010a); as probiotics induce host benets, in part at least, by modulation of the host microbiota it is pertinent to discuss the effect of probiotics on the gut microbiota of salmonids. The effect of probiotics on the gut microbiota of sh has been discussed in detail previously (Ring et al., 2005) but an update of literature is required. A wide range of probionts have been recovered from the digestive tract of salmonids after 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. and certain Gram-negative spp.) to salmonids may lead to high levels of colonisation and modulated GI microbial populations (Irianto and Austin, 2002b; Panigrahi et al., 2004, 2005; Aubin et al., 2005a; Kim and Austin, 2006a; Balczar et al., 2007a,b; Bagheri et al., 2008; Merrield et al., 2010a,b,c,d). Routinely, investigators enumerate probiont and total viable levels of the gut microbiota after feeding on supplemented diets but unfortunately few studies assess the composition of the indigenous microbiota. The main exceptions in salmonids are Gildberg et al. (1995), Aubin et al. (2005a,b), Wach et al. (2006) and Bagheri et al. (2008). Despite providing useful data and demonstrating broad level alterations on the gut microbiota previous studies do not present the full picture due to the culturedependent methods used. A plethora of culture-independent methods are available based on the sequence variability of the 16S and 23S rRNA genes that allow us to identify and quantify intestinal microbiota. Typically, when assessing the gut microbiota of sh DGGE remains the method of choice due to its rapid and inexpensive nature (Liu et al., 2008, Dimitroglou et al., 2009; Zhou et al., 2007, 2009). Indeed, Kim and Austin (2006a) demonstrated the usefulness of DGGE to assess the gastric longevity of carnobacteria strains fed to rainbow trout after reverting to non-supplemented diets. DGGE ngerprints revealed that Carnobacterium maltaromaticum and Carnobacterium divergens could survive in trout intestine for at least 3 weeks after dietary supplementation ended. The current literature provides a foundation, but our knowledge of the effect of probiotics on the gut microbiota of salmonids is limited because [1] most studies tend to be restricted to the enumeration of the probionts without investigation of the indigenous microbiota, [2] modern culture-independent molecular based techniques are not frequently utilised and [3] data is primarily restricted to rainbow trout. Future probiotic studies should incorporate PCR-DGGE techniques with subsequent 16S rRNA sequence analysis and/or microbial community diversity analysis, nMDS and similarity indices which have been used to study the gut microbiota of sh previously (Dimitroglou et al., 2009; Merrield et al., 2009). Furthermore, quantitative cultureindependent methods, such as FISH and qRT-PCR, should become a routine method for future investigations.
The histological effect of probiotics on the gut epithelium of salmonids has been assessed in vitro (Lvmo, 2007; Ring et al., 2007b; Salinas et al., 2008; Ring et al., 2010a). For example, Ring et al. (2007b) exposed the Atlantic salmon foregut to C. divergens, originally isolated from distal intestine of Atlantic salmon, as well as two well known pathogens: A. salmonicida and V. (L.) anguillarum. Light and electron microscopy demonstrated that pathogen-induced damage to the Atlantic salmon foregut could not be prevented or reversed, but could be marginally reduced in some cases. Despite such in vitro studies, in vivo data regarding the effects of probiotics on the intestinal morphology of salmonids is scarce. To the authors' knowledge, the only probiotic study which has demonstrated in vivo that probiotics may enhance the intestinal microvilli morphology of salmonids is that of Merrield et al. (2010d). Merrield and colleagues demonstrated in a preliminary study that dietary applications of P. acidilactici could signicantly improve microvilli length of the rainbow trout proximal intestine compared to the control group. However, microvilli density was not affected. The study also demonstrated that dietary treatments of B. subtilis + B licheniformis and E. faecium did not affect microvilli length or density. The reason for the lack of benets in the B. subtilis + B licheniformis and E. faecium was not elucidated but the authors speculated that it may be due to the perceived lack of mucosal colonisation (no colonisation by probiont-like cells) observed by electron microscopy in these groups. Instead, cells appeared to colonise the mucus layer. Further study is required to
Table 4 Summary of probionts that have been recovered from the digestive tract of salmonids after feeding on supplemented diets. Probiont Oncorhynchus mykiss Vibrio alginolyticus Vibrio uvialis Aeromonas hydrophila B. subtilis B. subtilis + B. licheniformis C .inhibens Carnobacterium spp. C. maltaromaticum Carnobacterium divergens P. acidilactici S. cerevisiae Leu. mesenteroides La. lactis L. sakei L. plantarum L. rhamnosus E. faecium Salmo salar Carnobacterium divergens C. inhibensb Salmo trutta Leu. mesenteroides La. lactis L. sakei Effect on indigenous microbiota assessed?a No No No No Yes No No No No Reference
Irianto and Austin (2002b) Irianto and Austin (2002b) Irianto and Austin (2002b) Newaj-Fyzul et al. (2007) Bagheri et al. (2008) Merrield et al. (2009abd) Irianto and Austin (2002b) Robertson et al. (2000) Irianto and Austin (2002b) Kim and Austin (2006a) Kim and Austin (2006a) Aubin et al. (2005a,b) Merrield et al. (2009c,d) Aubin et al. (2005a) Wach et al. (2006) Balczar et al. (2007b) Vendrell et al. (2008) Balczar et al. (2007b) Balczar et al. (2007b) Vendrell et al. (2008) Nikoskelainen et al. (2003) Panigrahi et al. (2005, 2005) Merrield et al. (2009abc) Gildberg et al. (1995) Jborn et al. (1997) Robertson et al. (2000) Balczar et al. (2007a) Balczar et al. (2007a) Balczar et al. (2007a)
3.6. Gastrointestinal morphology The endogenous gut microbiota inuence the development of the gut and are key components involved in the regulation of mucosal tolerance, development and differentiation (Rawls et al., 2004, 2007; Bates et al., 2006); however, maintenance of a healthy gut microbiota is likely to benecially affect the gut epithelial architecture at both the developmental and post developmental stage. Reducing the number of potential pathogens present within the GI tract may reduce mucosal damage and lead to improved absorptive surface area. Indeed, many sh pathogens can disrupt the integrity of the intestinal epithelium (Ring et al., 2007a,b; Salinas et al., 2008; Ring et al., 2010a).
Yes No Yes Yes No No No No No No No No Yes No No Yes Yes Yes
a Enumeration of TVC of indigenous levels or probiotics cells = No; identication of indigenous genera/species affected and/or use of alternative molecular methods for assessing changes in diversity, richness etc = Yes. b Carnobacterium strain K1 was later identied as C. inhibens.
understand these observations and assess the level of hostmicrobe interaction regarding the probionts investigated. 3.7. Nutrition Although many now consider probiotic applications in terms of health we must also remember that modulation of gut microbiota and enhanced gut morphology may help to improve the nutrition of the host. Modulation of the gut microbiota may enhance enzymatic activity (Wach et al., 2006) and the production of vitamins which are likely to bolster health and nutrition in the host. Indeed numerous studies have shown that the application of probiotics can improve feed conversion, growth rates and weight gain of sh including salmonids (e.g. Bogut et al., 1998, 2000; Taoka et al., 2006a; Wang et al., 2008b; Bagheri et al., 2008; ). For example, Bagheri et al. (2008) demonstrated that the application of B. subtilis + B. licheniformis could signicantly improve rainbow trout fry feed conversion ratio (FCR), specic growth rate (SGR), weight gain and protein efciency ratio (PER) after 2 months feeding on diets containing 3.8 109 CFU g 1. Furthermore, compared to the control group body protein composition was elevated and lipid levels decreased in the probiotic group. It is recommended that future studies monitor growth parameters and body composition. Beyond these parameters, little data exist with regards to other contributions to the digestive function. However, work by Aubin et al. (2005a) revealed that dietary P. acidilactici alleviated vertebral column compression syndrome (VCCS) in rainbow trout to a similar extent as antibiotics. The authors postulated that this conrmed that infection was the root cause of the syndrome and that probiotics were able to reduce disease prevalence, by either competing with gut pathogens or by stimulating favourable conditions in the intestine. This is a reasonable hypothesis, but in light of recent studies (Merrield et al., 2010c,d), we hypothesise that P. acidilactici may improve bone formation/mineralization via improved mineral uptake. Merrield et al. (2010c) observed a signicantly reduced condition-factor in rainbow trout fed P. acidilactici versus the control group; effectively increasing the length to weight ratio. Given that no other growth parameters had been affected in that study, it is clear that trout receiving probiotic treatments were in some way elongated or that the control group were to some extent stunted. The proposed mode of action of probiotics acidifying the gastric environment may have played a signicant part in the solubilization and thus uptake of minerals from the digestive tract. Indeed, it is known that acidication, leading to reduction and thus higher solubility of minerals, increases the uptake of many metal ions. This is most notably demonstrated as ascorbic acid reduces iron to its more soluble form: the reason that vitamin C is associated with a higher iron uptake efciency (Fairweather-Tait, 1995). In the case of probiotic treatments, this acidication is achieved through the production of lactic acid and short-chain fatty acids. Evidence in higher vertebrates supports this suggestion: in humans, low serum values of magnesium, copper and zinc are associated with osteoporosis in post-menopausal women (Scholz-Ahrens et al., 2007), a clear correlation of mineral assimilation/circulation and correct bone formation. In their review, Scholz-Ahrens et al. (2007) also highlighted rat-studies showing that prebiotics stimulated the absorption of iron and of bone-relevant minerals such as calcium, magnesium and zinc. Few studies have assessed the effects of probiotics or synbiotics on metabolism of minerals or subsequent effects on bone health; yet it has been suggested that there is an independent probiotic effect on facilitating mineral absorption (Scholz-Ahrens et al., 2007). Using transmission electron microscopy (TEM) Merrield et al. (2010d) observed increased enterocyte endocytic activity and increased microvilli length in rainbow trout fed dietary P. acidilactici. This may support the present hypothesis that an increased uptake of minerals/calcium may occur in trout fed P. acidilactici. However,
further studies are required to conrm this speculative hypothesis. There appears to be a growing volume of literature demonstrating the benecial role of probiotics in the uptake of minerals from the gut, and subsequent deposition in the bone. However, a further possibility also exists to explain how probiotics can improve mineral uptake from the intestinal lumen. It has been proposed that the bacterial activity in the gut is able to degrade the mineral-complexing phytate (ScholzAhrens et al., 2007). Whilst aquaculture diets tend to be supplemented with phosphorus to ensure that adequate free-phosphorus is available in the ration, little attention is paid to the impact of phytic acid on other minerals. In order to further explore this avenue, it is proposed that trials be conducted looking at the impact of supplemental phytase enzyme on the bone development in farmed salmonids. 3.8. Feeding strategies Many studies have now provided proof of concept that probiotics can be effective for use within aquaculture but probiotic application methods have varied greatly and are not always practical for production level sh farming. If a salmonid sh farmer decides to use probiotics how should the application be conducted? We must consider: [1] the probiont, [2] supplementation form, [3] vector of administration, [4] dosage level and [5] duration of application. 3.8.1. Choosing a probiont Bacillus spp., LAB, certan Gram-negative spp. and yeast have all demonstrated benets for salmonids (refer to Table 1). Initially, it was suggested that probiotic bacteria could be host-specic with their effects limited to their natural hosts (Fuller, 1973; Myr-Mkinen et al., 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 specic probiont will likely be dependent on the sh species, rearing conditions and desired outcome of supplementation (i.e. immuno-stimulation, disease prevention, improved growth performance etc). 3.8.2. Supplementation form Most commonly in salmonid studies, live-cultures are sprayed or top-dressed onto basal diets (e.g. Panigrahi et al., 2005, Balczar et al., 2007a,b; Vendrell et al., 2008; Merrield et al., 2010a,b,c,d) but freeze-dried/lyophilised cells (e.g. Aubin et al., 2005a; Panigrahi et al., 2005, 2007; Merrield et al., 2010c), dead cells (e.g. Irianto and Austin, 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; NewajFyzul et al., 2007) and spores (e.g. Raida et al., 2003; Bagheri et al., 2008) have all showed some degree of success. With regards to applications at the farm level it may be more practical to use dead cells, lyophilised cells or spores rather than culturing live cells. Few studies have compared both live and dead probiotic applications in sh but studies have demonstrated that probiotic viability can be a factor (Brunt and Austin, 2005; Panigrahi et al., 2005; Taoka et al., 2006b). Panigrahi et al. (2005) concluded from their study, that the potential benets of heat-killed cells should not be overlooked but that viable forms induced better results. Furthermore, as no differences between the live-sprayed and freeze-dried forms were observed in the investigation, Panigrahi et al. (2005) suggested the freeze-dried administration method merited further consideration as a practical mode of delivery for aquaculture practices. One of the benecial attributes of Bacillus species for applications as probiotics is their spore-forming abilities which allows for greater viability after pelleting and high resistance to gastric conditions (Hyronimus et al., 2000; Casula and Cutting, 2002; Hong et al., 2005). Furthermore, the application of dietary Bacillus spores has proved effective in probiotic applications for trout (Raida et al., 2003; Bagheri et al., 2008). As it is
10
not practical to mass culture cells prior to application at the farm perhaps lyophilised cells or spores may be a more practical option. 3.8.3. Vector of administration Most commonly probiotics have been administered via the feed; however, some information regarding administration of probiotics to salmonids via rearing water is available. Such application has proved both effective and ineffective at reducing mortalities (refer to Table 3). There is limited data comparing the effectiveness of water based supplementation compared to oral administration in salmonids, but water based applications are likely to be limited in ow-through rearing systems and sea/lake cages. These methods may be more applicable in re-circulation facilities or for bathing treatments applied regularly or during times of disease. Furthermore, data regarding water-administered probionts to salmonids appear to be available only with regards to disease resistance, which leaves many questions regarding the efcacy to other host related parameters. 3.8.4. Dosage level Dose can be important as differences of host response between different dietary probiotic levels have been observed (Nikoskelainen et al., 2001, 2003; Panigrahi et al., 2004; Bagheri et al., 2008). Panigrahi et al. (2004) investigated the potential doseresponse relationship of rainbow trout with L. rhamnosus. Panigrahi et al. (2004) assessed the cellular and humoral immune response of rainbow trout fed diets containing L. rhamnosus at either 109 or 1011 CFU g 1 for 30 days. After 30 days feeding, both probiotic groups displayed signicantly higher HK leukocyte phagocytic activity but only the high level group displayed signicantly improved serum lysozyme and alternative complement activity compared to the control group. High LAB levels were recovered from the GI tract throughout the supplemented feeding period but LAB levels in the intestine also appeared to be dose dependent. Dose dependent studies are currently limited and somewhat contradictory. Further studies are required, in particular with salmon, before guideline levels can be suggested with any degree of condence. Appropriate levels are likely to vary depending on the probiont species, host sh species, host physiological status, rearing conditions and the specic goal of the feeding application (i.e. health, disease resistance, nutrition etc). 3.8.5. Supplementation duration Studies have assessed potential probiotic applications for periods as short as 6 days (Jborn et al., 1997) and for periods of up to 5 months (Aubin et al., 2005a). In some rare cases, the inuence of probiotic feeding duration on treatment efcacy has been studied (Panigrahi et al., 2005; Balczar et al., 2007a; Sharifuzzaman and Austin, 2009). However, information on long-term efcacy is not available. Supplementation has proved to provide short-term benets but generally probionts have not been detected within the GI tract for periods beyond one to three weeks after reverting to non-supplemented diets (Robertson et al., 2000; Balczar et al., 2007a; Panigrahi et al., 2005; Kim and Austin, 2006a) and presumably probiotic benets are lost after the probiont is removed from the host. Therefore there appear to be 3 distinct options for administrative strategy: [i] short-term application conned to times of need, [ii] constant supplemented feeding or [iii] cyclic feed of supplemented diets. [i] Short-term supplementation has proved to be effective at gastric colonisation, stimulating the immune system and providing protection 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). Such studies provide important proof of concept that the use of selected probiotics can be an effective method for protection against disease; however, a sh farmer cannot predict when the onset of disease may
occur in order to provide probiotic feeding in the weeks prior to infection. Will treatment be effective after the onset of disease has been detected? Further work is required to answer this question. Restricted feeding to times conducive of stress may be benecial yet currently there is little data to support this strategy. [ii] Constant supply of probiotics, incorporated into the diet as a feed supplement, may provide benets but there is little data available for the long-term use of probiotics. However, the study by Aubin et al. (2005a) provides some useful data regarding long-term applications. Aubin et al. (2005a) compared probiotic recovery levels over time and observed that levels were higher after 20 days (with P. acidilactici levels of log 2.5 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. cerevisiae were not detected). When considering long-term use of probiotics it is worth considering that the immune response is often diminished with long-term use of immuno-stimulants which often leads to the immune status reverting back to control levels or in extreme cases may lead to immunosuppression (Sakai, 1999; Smith et al., 2003; Bricknell and Dalmo, 2005). Although it is not currently clear whether this is the case with long-term application of probiotics, we must consider the possibility that it may not be pertinent to use constant probiotic supplementation for extended periods. [iii] Short-term-cyclic (termed pulse-administration by Bricknell and Dalmo, 2005) probiotic feeding strategies may be benecial, as is the case of some immuno-stimulant products, although there is no data currently available to support or disprove this hypothesis. Such a strategy could involve feeding probiotic supplemented diets and unsupplemented diets alternately for short periods (e.g. for periods of 2 or 4 weeks) cyclically. Application in this way may provide the direct benets of short-term application during the supplemental feeding phase and during the un-supplemented stage where gastric probiotic populations persist for a number of weeks (Balczar et al., 2007a; Kim and Austin, 2006a) may provide a level of protection against transient pathogens and could continue to induce some degree of immunostimulation (Nikoskelainen et al., 2003; Balczar et al., 2007a). This strategy may help to avoid over-stimulating the immune response whilst maintaining a level of protection/immuno-stimulation. Currently there is no data to support this hypothesis and future research should consider this application strategy. Current research provides proof of the probiotic concept for salmonids but applications within these studies are so varied and often impractical for industrial scale farming that it is difcult to plan an effective feeding strategy for commercial level applications. Future work, including practical evaluation under farming conditions, once safety concerns have been considered, should focus on providing a remedy to this. 3.9. Issues with industrial level scale-up Following on from the aforementioned problems and lack of knowledge pertaining to applications of feeding strategies mentioned in the previous section, several other issues have, and continue to, hamper the transition from scientic trials to industrial scale applications. Several studies have been conducted at sh farms (refer to Table 1) but industrial level applications on mass are not frequently reported. It is likely however, that industrial scale applications are relatively common, as commercial bodies wish to assess their products but due to issues regarding intellectual property rights such data is conned to industrial stakeholders and not widely shared with the academic community until sometime later. It is important to mention that in all cases, at least within the European Union, each eld trial involving feed additives not yet authorised require approvals by national food safety association equivalents; making industrial level applications on mass difcult. From industrial and commercial perspectives, such trials are of course essential to demonstrate the efcacy of probiotics at the farm level
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and to evaluate the nancial outcome of using such products in the eld. Furthermore these trials also demonstrate the feasibility of the probiotic application form a practical point of view (conditions of use, stability under different storage conditions etc). Indeed, in order to be used successfully at an industrial scale, technological issues concerning probiotic incorporation into extruded feeds must be considered because as discussed previously, in many cases probiotic viability impacts effectiveness. Thus probiotic microorganisms must survive the stressful conditions of feed processing and storage which is a particular concern in aquaculture since aquafeed processing conditions are physically intensive compared to terrestrial animal feeds. Therefore, probiotic incorporation in aquafeeds remains limited at industrial levels, particularly due to the drawback of heat stability. In order to overcome such issues, novel approaches in application are required. Providing probiotic living cells in a protected form with a physical barrier against adverse environmental conditions is a technique currently receiving considerable interest. For instance, microencapsulation of probiotic bacterial cells has been developed (Kailasapathy, 2002) and patented microencapsulated bacteria or yeasts are today available on the market (Ziggers, 2005). Recently dramatic improvement in this technology has been achieved by yeast and bacteria producing companies and some active yeast commercial products have even been reported to resist temperatures up to 90 C (M. Castex, pers. communication). However, the current microencapsulation technology is not yet sufcient to protect live bacteria in the case of extruded feeds. While developing new microencapsulation technologies, the only alternative for now is to develop alternative solutions with the feed manufacturers in order to apply probiotics on their feed products. Several systems exist and the post-pelleting spraying in oil or water is generally the best option. For instance this procedure applied to salmon feeds has shown efcient results in terms of homogeneity and conformity to the expected dosage at both laboratory and industrial levels (Ziggers, 2005). Furthermore, after following the required procedure for pressure, temperature, ow rate etc, probiotic survival remains stable on pellets for a few months. However, the implementation of post-pelleting systems on feed plants generally requires signicant investments, including specic equipments such as screw coater and probiotic injection units, and can only be applied after the efcacy of the concerned probiotic has been fully validated in the eld. In the future of probiotic development at an industrial scale, alliance and transversal experiences between biological technology and industrial processing must be conducted in order to offer innovative and easy to use solutions for the feed industry. Finally, one of the main stumbling blocks from a practical point of view, in order to be used at a large scale, is that candidate probiotic species must satisfy stringent regulations. Indeed, safety considerations are increasingly taken into account for the development of probiotics. As an example, the European Union regulates the authorisation, marketing and use of probiotics as feed additives under the European Parliament and Council Regulation (EC) No (1831). In accordance with this Regulation, the Commission, having rst consulted the European Food Safety Authority (EFSA), has established rules (Commission Regulation (EC) No 429/ (2008)) concerning the preparation and the presentation of applications. Then the registration of a probiotic requires the preparation of a dossier with data and studies demonstrating efcacy and safety of the product for animals, consumer and environment. This safety concerns are of utmost importance in aquaculture since, as mentioned by Wang et al. (2008b), new species and specic strains identied as potential probiotics for aquatic species do not share the historical safety of traditional or widely tested strains such as LABs (Adams, 1999). However, a long-term Research & Development project, initiated from 2001 through the OFIMER program with BioMar and several research institutions, has resulted in the rst EU-approval of the use of probiotics for 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 and will result in the introduction of the rst approved industrial trout and salmon feed containing probiotics in 2010 (M. Autin, pers. communication). 3.10. Future probiotic work Scientists working on the topic of probiotics should also focus their research on the use of other molecular methods than DGGE. As little information is available on quorum sensing, different staining methods (e.g. immunogold, FISH and green uorescent protein labelled bacteria) should be utilised to investigate adherence and colonisation of probiotic and pathogenic bacteria and their interactions within the sh digestive tract. Scientists might protably investigate the interactions between probiotics and pathogens in the digestive tract of sh as suggested by Ring et al. (2010a). In addition to molecular techniques to evaluate the microbial community as a result of probiotic supplementation, important endogenous bacteria isolated by traditional cultivation should be investigated for their metabolic capabilities such as degradation of anti-nutrients, as it has been shown that LAB fermentation can improve the nutritional value of soybean meal (Refstie et al., 2005). This is highly relevant to investigate as one of the main challenges in aquaculture is the use of plant protein sources in the diets (Gatlin et al., 2007). Moreover, adhesion is one of the most important selection criteria for probiotic bacteria because it is considered a prerequisite for colonisation. Although some information is available regarding probiotic adhesion to and growth within sh intestinal mucus, it is essential that probiontmucosal interactions be assessed in future studies. It is at present not clear what role the gut microbiota, or indeed probiotics, play in mediating salmonid gastric development, gene expression and mucosal tolerance. Thus, much of our understanding regarding effects in salmonids, and sh in the broad sense, come from studies with higher vertebrates. However, Kim and Austin (2006b) assessed the effects of Carnobacterium on gene expression of gut cells and HK leukocytes in vitro by co-culturing with the probiotic cells and concluded that although the probiotics did not signicantly induce cytokine mRNA in gut cells, the probiotics could stimulate innate immunity as demonstrated by the expression ratios of IL-1 and TNF of HK cells. The study provides a platform for our understanding of the effects of probiotics on the expression of cytokines in gut cells but further in vivo work is required to provide a more broad understanding of the complex interactions of probiotics and the host mucosa. Future work should aim to enhance our understanding of these interactions by isolating gut cells of probiotic fed salmonids and assessing the expression of immune activatory or immunoregulatory cytokines (e.g. IL-1, IL-8 and TNF and INF), pattern recognition receptors (eg. TLRs and scavenger receptors) and anti-microbial proteins (e.g. IRF3, INF type 1, Mx 1, TLR3, TLR7, TLR9, NOS2 and GBP). Such studies will elucidate the cellular and molecular mechanisms involved in probiotic modulation of the mucosal immune system, informing future approaches in aquaculture in the maintenance of an efcient immune system which is reected in sh quality and productivity. 4. Prebiotics The use of probiotics in many cases, as discussed previously, may be difcult in commercial aquaculture because of the low viability of the bacteria after pelleting and during storage, leaching from the feed particle in rearing water, as well as problems related with feed handling and preparation. As an alternative (or also considered for use in tandem: synbiotics), prebiotics have been assessed in an attempt to overcome issues associated with probiotic applications. From an endothermic point of view, a prebiotic is dened as a non-digestible food ingredient that benecially effects the host by selectively stimulating the growth and/or the activity of specic health
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D.L. Merrield et al. / Aquaculture 302 (2010) 118 Table 5 Summary of potential prebiotic applications for salmonids. Species Prebiotic Parameters investigated GM, GH GP, FU, GH GP, FU, IR BC, GP, IR, DR References Dimitroglou et al. (2009)c Yilmaz et al. (2007)a Staykov et al. (2007)a Rodriguez-Estrada et al. (2009)a Sealey et al. (2007)a Dimitroglou et al. (unpublished data)c Grisdale-Helland et al. (2008)a Refstie et al. (2006)a Bakke-McKellep et al. (2007)a Olsen et al. (2001)a Ring et al. (2006)a
promoting bacteria that can improve the host health (Gibson and Roberfroid, 1995). Prebiotics mainly consist of oligosaccharides promoting benecial bacterial growth within the GI tract (Yazawa et al., 1978; Gibson et al., 2003). Readers with specic interest into the classication and types of carbohydrates with potential to be classed as prebiotics are referred to the reviews of Gibson et al. (2004) and Roberfroid (2007). In a review for human intestinal microbiota the prebiotic denition was updated. Gibson et al. (2004) suggested that a prebiotic has to: resist gastric acidity, hydrolysis by (mammalian) enzymes and GI absorption. be fermented by the intestinal microbiota. stimulate selectively the growth and/or activity of intestinal bacteria associated with health and well-being. According to the authors, each one of these criteria is important but the third criterion, concerning the selective stimulation of growth and/or activity of benecial bacteria, is the most important and the most difcult to achieve. This is especially true because this step requires anaerobic sampling and reliable quantitative microbial analysis should be undertaken for a wide range of bacterial species (i.e. total aerobic and anaerobic bacteria as well as total counts of Bidobacteria, Enterobacteria, Lactobacillus, etc). According to Gibson et al. (2004), only three oligosaccharides were classied as prebiotics: inulin, transgalactooligosaccharide (TOS) and lactulose. A more recent study includes fructooligosaccharides (FOS) in the list of prebiotics (Roberfroid, 2007). Due to the limited data available regarding sh, the carbohydrates that have fullled the requirements, or have shown potential to full the requirements, of the denition of prebiotic in the aforementioned reviews will be discussed in context to salmonids. Prebiotics have demonstrated some benets in sh (Burr et al., 2005; Gatlin et al., 2006; Ring and Olsen, 2008; Ring et al., 2010b) but the use of prebiotics in salmonid studies remains relatively limited (Table 5). Based on the prebiotic results available per se, Ring et al. (2010b) concluded in their review that to fully conclude on the effects of adding prebiotics in sh diets, more research efforts are needed in order to provide the aquaculture industry, the scientic community, the regulatory bodies and the general public with the necessary information and tools. 4.1. Mannanoligisaccharides (MOS) MOS are glucomannoprotein-complexes derived from the cell wall of yeast (S. cerevisiae) (Sohn et al., 2000). Its use in terrestrial animals is well documented (for a review see Benites et al., 2008; Klebaniuk et al., 2008; Yang et al., 2009) but the use of MOS has recently been introduced in salmonid culture also (Staykov et al., 2007; GrisdaleHelland 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 MOS supplementation on on-growing Atlantic salmon. Dietary MOS was supplemented at 1% to salmon for a period of 16 weeks. The results showed that apparent energy digestibility of the MOS supplemented diet was increased compared with the control treatment. Analysis of body composition revealed that gross energy content was increased with the addition of MOS but crude protein was reduced. Furthermore, whole blood neutrophil oxidative radical production and serum lysozyme activity were reduced in sh fed the MOS supplemented diet. In the rainbow trout study by Staykov et al. (2007), growth performance and immune parameters of sh reared either in fresh water net cages or fresh water raceways were investigated. Compared to the control sh, 0.2% dietary MOS supplementation increased nal body weight, reduced FCR and mortalities in both net cage and raceway reared trout. MOS supplementation induced signicant improvements of immune parameters compared to the control
Oncorhynchus MOS MOS mykiss MOS MOS
Salmo salar
GroBiotic A* BC, DR, GP, IR MOS GP, FU, GH, RP MOS, GOS, FOS Inulin Inulin Inulin Inulin GP, FU, BC DE, OI, PC, GH BP, OI, GM, GH GH GM
Salvelinus alpinus
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 organosomatic indices, PC plasma chemistry, BP body parameters. a In vivo studies. b In vitro experiments. c Field experiments. * GroBiotic-A is a mixture of partially autolyzed brewers yeast, dairy ingredient components and dried fermentation products.
groups. However, variations in the benets were observed between net cage reared trout and raceway reared trout. Serum antibody titre, bactericidal activity and lysozyme activity were signicantly increased in the net cage reared trout; in the raceway reared trout, MOS supplementation signicantly increased serum lysozyme activity, classical and alternative pathway complement activity. In another rainbow trout study, the effect of dietary MOS (1.5%, 3.0% and 4.5%) on growth performance and body composition was investigated by Yilmaz et al. (2007). The authors reported increased growth performance when the sh were fed the lowest (1.5%) MOS level. Conversely, carcass protein content increased with increasing MOS supplementation. Histological analysis of the proximal intestine showed that sh fed 1.5% and 3.0% MOS displayed longer villi than both the control group and the 4.5% MOS group. However, FCR, PER and hepatosomatic index (HSI) remained unaffected. In an in vivo study on rainbow trout ngerlings fed 0.4% MOS for 12 weeks, Rodriguez-Estrada et al. (2009) reported that inclusion of MOS to the diet, stimulated growth, haemolytic- and phagocytic activity, mucosa weight and improved survival when the sh were challenged with V. (L.) anguillarum. The results from a recent study with juvenile (initial weight 38 g) and sub-adult (initial weight 112 g) rainbow trout reared under commercial farming conditions demonstrated that dietary MOS had a clear effect on both the intestinal microbiota and histology (Dimitroglou et al., 2009). Supplementation of 0.2% MOS to the diet signicantly reduced the aerobic culturable bacterial load within the GI tract. Additionally, changes in the relative abundance of the microbiota were observed. In the juvenile sh group, MOS fed trout displayed a signicant reduction of both the relative and absolute abundance of Micrococcus spp., Aeromonas/Vibrio spp. and a group of unidentied Gram-positive rods. Coinciding with these changes, a signicant increase of the relative abundance of enterococci was observed. Compared to the control group, the MOS fed sub-adult trout displayed a signicant reduction of Micrococcus spp. and Enterobacteriaceae and an increase of Pseudomonas spp. Culture-independent analysis using PCR-DGGE showed that dietary MOS supplementation resulted in reduced bacterial species richness in both juvenile and sub-adult groups compared to the respective controls. Non-metric multidimensional scaling (nMDS) analysis also demonstrated a clear
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MOS induced effect on the microbiota; dietary MOS caused a distinct spatial shift of bacterial communities away from the control groups. Histological evaluation using light microscopy revealed that the subadult MOS group displayed signicantly increased intestinal absorptive surface in both the proximal and distal intestinal regions. Furthermore, electron microscopy revealed increased microvilli density in the distal intestinal region and increased microvilli length in both intestinal regions in the sub-adult MOS fed trout. 4.2. Inulin Inulin is a term applied to a heterogeneous blend of fructose polymers differing from FOS (or oligofructose), a subgroup of Inulin, by the degree of polymerisation (DP): inulin DP N 10 and FOS DP 10 (Niness, 1999). Inulin is found naturally in a variety of plant foods such as bananas, barley, chicory, garlic, Jerusalem artichoke, leeks, onions and wheat (Roberfroid, 1993; van Loo et al., 1995) and its use in endothermic animals is well documented in several comprehensive reviews (Havenaar et al., 1999; Gibson et al., 2004; Possemiers et al., 2009; Kelly, 2009; Wang, 2009). The rst study using inulin in salmonids was conducted by Olsen et al. (2001), who fed high level (15%) dietary inulin to on-growing Artic charr. After 4 weeks of feeding, histological analysis revealed that inulin caused destructive effects in the lay-out and structure of microvilli in the distal intestine. Additionally, lamellar bodies dominated the enterocytes interior and the presence of vacuoles also increased. Pyloric caeca were also examined and the effect of inulin was restricted only in the lysosomal bodies which were increased from 0.5% to 2.2% in the inulin fed sh. Some information is available about inulin fermentation by lactic acid bacteria isolated from the sh gut, notably, C. maltaromaticum, Carnobacterium mobile and Carnobacterium spp. (Ring, 2004). Based on this nding, a subsequent investigation of the effect of 15% dietary inulin on the culturable aerobic and facultative aerobic autochthonous microbiota of the distal intestine of Arctic charr was conducted using 16S rRNA identication, assessment of isolates ability to utilise inulin and electron microscopy (Ring et al., 2006). Substituting dietary dextrin with inulin reduced the bacterial population and also altered microbial composition. The general ndings from this study were that the distal gut microbiota of control sh fed (15% dextrin) contained Pseudomonas spp., Psychrobacter glacincola, C. divergens. Micrococcus spp., Staphylococcus spp. and Streptococcus spp. while the gut microbiota of sh 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 carnobacteria as some strains of both Staphylococcus spp. and Streptococcus spp. isolated from the control group also had this ability. Although changes in the microbiota were observed, no clear conclusion can be given that inulin stimulates colonisation of inulin utilising bacteria in the gut of Arctic charr based on the results of Ring et al. (2006). Electron microscopical analysis of distal intestine conrmed the results of traditional culture-based microbial analysis as fewer bacterial cells were observed between microvilli and associated with the surfaces of enterocytes of sh fed inulin compared to that for the dextrin fed sh. Refstie et al. (2006) conducted a study to investigate the effect of dietary inulin (7.5%), with and without oxytetracycline addition (0.3%), on Atlantic salmon for a 3 week period. At the end of the trial blood plasma analysis revealed that the concentration of free fatty acids, glucose, cholesterol and triacylglycerides were not affected by the dietary inulin or the oxytetracyline supplementation. Fish fed inulin displayed a higher relative gut weight (relative to total body weight) but relative liver and stomach weights were unaffected. Histology of the distal intestine revealed that neither inulin nor oxytetracycline induced any morphological changes. Additionally, analysis of GI tract for trypsin and amylase activity and brush border membrane alkaline phosphatase and leucine aminopeptidase activity
revealed that dietary inulin signicantly affected the relative trypsin activity in the distal intestine, the total amylase activity in the proximal and the distal intestine and the leucine aminopeptidase activity of the distal intestine (expressed relative to protein). Apparent amino acid absorption was not affected by inulin supplementation. A more recent study with a similar experimental design, dietary supplements and trial duration to that of Refstie et al. (2006) was conducted by Bakke-McKellep et al. (2007). The results revealed that inulin, with or without oxytetracycline addition did not affect nal body weight or body length. With the exception of the distal intestinal somatic index no other gastric organosomatic indices were affected. Histological evaluation of the proximal and the distal intestine revealed that neither inulin or oxytetracycline inuenced the intestinal morphology. However, the muscle layer of the proximal intestine of inulin fed sh showed moderate leucocytic cell inltration (in 6 out of 12 sh) compared with the control sh. Immunohistochemical measurements of proliferating cell nuclear antigen demonstrated that inulin supplementation did not affect the positive proliferative compartment length. However, inulin with oxytetracycline addition reduced the proliferative compartment length. Additionally, microbial enumeration of the gut microbiota revealed that inulin reduced the allochthonous viable bacteria in digesta of both the proximal and distal intestine but autochthonous population levels were unaffected. The contrasting ndings in gut morphology in the study with Arctic charr (Olsen et al., 2001) compared to that reported in Atlantic salmon (Bakke-McKellep et al., 2007) may be due to dietary levels of inulin, 15% for Arctic charr vs. 7.5% for Atlantic salmon, or different analytic methods; transmission electron microscopy (TEM) vs. light microscopy (LM). LM used by Bakke-McKellep et al. (2007) does not allow sufcient resolution to evaluate changes in the organization of microvilli and the presence of intracellular lamellar bodies in distal intestinal enterocytes as observed by TEM (Olsen et al., 2001). Based on the results from these two studies we highly recommend that both LM and TEM are included in future studies. 4.3. Other prebiotics The commercial product GroBiotic-A is a mixture of partially autolyzed brewers yeast, dairy ingredient components and dried fermentation products (Li and Gatlin 2005). An overview of the studies carried out using GroBiotic-A in aquaculture is presented by Ring et al. (2010b), but to our knowledge only one study has been conducted on salmonids. Sealey et al. (2007) described the effect of dietary partially autolysed yeast and GroBiotic-A on rainbow trout. Fingerlings were fed the experimental diets containing 2% prebiotic for 9 weeks. The results showed that growth performance, immune responses and TNF- mRNA expression level remained unaffected throughout the experimental period. On the contrary, whole body energy content and survival from infectious hematopoietic necrosis virus (IHNV) challenge was signicantly increased in sh fed either partially 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 the effect of 1% dietary FOS supplementation on on-growing Atlantic salmon after feeding for 16 weeks. The results demonstrated that FOS fed salmon displayed some improvement of feed efciency ratio (FER) compared to the control group. However, nal body weight, apparent nutrient digestibility and carcass proximate analysis remained unaffected. Additionally, whole blood neutrophil oxidative radical production and serum lysozyme activity remained unaffected. In this study, GrisdaleHelland et al. (2008) also included a treatment of dietary galactooligosaccharides (GOS) which, to our knowledge, is the only GOS study conducted with salmonids (Grisdale-Helland et al., 2008). Using the same experimental protocols, on-growing Atlantic salmon fed 1% dietary
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prebiotic for 16 weeks, Grisdale-Helland and colleagues observed that GOS was unable to affect growth parameters relative to the control group. Apparent nutrient digestibility and body composition also remained unaffected. However, body gross energy content of GOS fed sh was reduced. Whole blood neutrophil oxidative radical production and serum lysozyme activity remained unaffected with GOS incorporation in the diet. Readers with special interest in the different aspects on the use of prebiotics (MOS, FOS, inulin, short-chainFOS, GOS, xylooligosaccharides, arabinoxylooligosaccharides isomaltooligosaccharides) in other sh species are referred to the recent review of Ring et al. (2010b) and the results of El-Dakar et al. (2007), Burr et al. (2008, 2009) and Lochmann et al. (2009).
6. Concluding remarks and future perspectives Current research provides a foundation but applications within these studies are often impractical at industrial level farming that it is difcult to plan feeding strategies for commercial level applications. Future efforts must focus on implementing more practical applications as well as scientic studies designed to understand the mechanisms that underpin and mediate the observed host benets. In this context, growth performance parameters and body composition analysis should be incorporated into future trials. Additionally, digestibility and costbenet analysis should be considered; this may be of particular relevance if selected probiont candidates display key digestive enzymatic attributes with regards to selected dietary components (plant proteins, chitin etc) which can then be supplemented to diets containing high levels of host non-digestible material. Although many tend to think of biotic applications in terms of health and disease benets we must not overlook the benets in terms of aiding host digestive function. Considering the gaps in the current knowledge, future investigations of biotic applications for salmonids should expand to more studies on Atlantic salmon and other important salmonids (e.g. Arctic charr, Chinook salmon Oncorhynchus tshawytscha, coho salmon Oncorhynchus kisutch, brown trout and sea trout S. trutta morpha trutta) as present data is heavily based on rainbow trout. Likewise, research at the larval stage, a critical time in the life cycle, should also be a focal point of concerted interest as most salmonid probiotic studies are based on juveniles. The application of probiotics has been demonstrated to improve tolerance of other sh species to environmental stressors, particularly at the 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 authors knowledge there is currently no literature available regarding the effect of biotics on salmonids during stress (stress associated with bacterial challenge studies not withstanding). It is at these critical windows that the role of biotic dietary supplements may offer an effective barrier against pathogenic invasion and subsequent mortalities; future studies are required in order to see if such benets can be achieved with salmonids. Acknowledgements The authors, as well as their respective institutions, would like to dedicate this article to their dear departed colleague and friend, Bruno Rochet (who died on 5 November 2009 at the age of 55 years). Bruno was the business development director for Lallemand Animal Nutrition, having joined Lallemand in 1998, after numerous years of working on the use of probiotics in Animal Nutrition. He was the founder and rst president of the European Probiotic Association established in 1999. As one of the pioneers in the eld, the recent European demarche and pursuit of the authorisation and use of probiotics in aquaculture could be attributed to his vision and foresight. In the last ve years, he had pursued his passion on aquaculture with determined dedication, initiating various research and developmental projects in the area and, in particular, the use of probiotics in aquaculture. We will all miss this very unique, passionate and dedicated gentleman who has greatly contributed to the use of probiotics in aquaculture and of course, the development of the European probiotics business in general. References
Adams, M.R., 1999. Safety of industrial lactic acid bacteria. J. Biotechnol. 68 (171), 178. Andlid, T., Vzquez-Jurez, R., Gustafsson, L., 1995. Yeast colonizing the intestine of rainbow trout (Salmo gairdneri) and turbot (Scophtalmus maximus). Microb. Ecol. 30, 321334. Arijo, S., Brunt, J., Chabrilln, M., Daz-Rosales, P., Austin, B., 2008. Subcellular components of Vibrio harveyi and probiotics induce immune responses in rainbow trout, Oncorhynchus mykiss (Walbaum), against V. harveyi. J. Fish Dis. 31, 579590.
4.4. Future prebiotic work Three of the gold standards to be included in future prebiotic studies are the effect on immune responses, gut microbiota and challenge studies. Very few studies have assessed the effects of prebiotics on the immune response of salmonid sh (Sealey et al., 2007; Staykov et al., 2007; Rodriguez-Estrada et al., 2009). Based on this fact we suggest that a full understanding of the uses of prebiotics will only be obtained by more intensive studies investigating the effects of prebiotics, in modulating both rapid-acting innate responses and slower pathogen-specic adaptive immune responses, in the context of both soluble and cellular effectors relevant to the gut mucosa. This will only adequately be addressed in comparative studies of healthy and pathogen challenged sh models. In the 70s, 80s and 90s, the majority of investigations of the intestinal microbial communities of sh focused on culturable aerobic populations (for review see Cahill, 1990; Ring et al., 1995; Ring and Birkbeck, 1999). These investigations can be useful for determining the dominant aerobic and facultative anaerobic bacteria, but are not appropriate for isolating strict anaerobic bacteria. Even when several selective growth media are used culturable studies still do not present a correct overview of the gut microbiota. Therefore we recommend that molecular methods such as DGGE and FISH are used in future prebiotic studies. To our knowledge, very few studies have used DGGE to evaluate the effect of prebiotics on gut microbial community in sh and shrimp (Li et 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 also be 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 competitive advantage (a fermentable energy source) over competing endogenous populations; Thus, effectively improving the survival and implantation of the live microbial dietary supplement in the gastrointestinal tract of the host (Gibson and Roberfroid, 1995). To the authors knowledge only one synbiotic study has been conducted in salmonids (Rodriguez-Estrada et al., 2009). In this study the individual application of the dietary E. faecalis and MOS provided a wide range of benets with regards to immune response and survival in a challenge study with V. (L.) anguillarum. However, it was noted that synbiotic feeding (E. faecalis + MOS) yielded signicantly better results than either individual probiotic or prebiotic application. Compared to the control, probiotic feeding failed to inuence growth performance but MOS however signicantly elevated weight gain, SGR and FCR. Interestingly, these parameters were further elevated in sh fed the synbiotic diet. In order to clarify similar effects on other salmonids we recommend that further synbiotic studies should be conducted.
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Contents lists available at ScienceDirect

D.L. Merrield et al. / Aquaculture 302 (2010) 118

D.L. Merrield et al. / Aquaculture 302 (2010) 118

D.L. Merrield et al. / Aquaculture 302 (2010) 118

D.L. Merrield et al. / Aquaculture 302 (2010) 118

Bacterial kidney disease Winter ulcer

Renibacterium salmoninarum Moritella viscosa

Evenden et al. (1993) Benediktsdottir et al. (2000)

S. iniae La. garvieae

A. sobria, Bacillus spp. A. sobria

IP CO O. mykiss IP IM, CO, IP

Yes Yes Yes Yes

D.L. Merrield et al. / Aquaculture 302 (2010) 118

V. anguillarum V. anguillarum V. anguillarum V. anguillarum

CO CO CO S. salar CO CO CO IM in vitro in vitro S. trutta Asymptomatic carrier O. mykiss IM IM IP IP

IP IP S. salar CO IM in vitro CO IM O. mykiss IP IP CO IM IP

Fin rot White spot B. subtilis

S. salar CO O. mykiss ID O. mykiss IM

C. inhibensa A. sobria, Brochothrix thermosphacta A. sobria, Brochothrix thermosphacta

Y. ruckeri A. bestiarum Ichthyophthirius multiliis Aeromonas sp.

D.L. Merrield et al. / Aquaculture 302 (2010) 118

Yes No Yes Yes No No No No No No No No Yes No No Yes Yes Yes

D.L. Merrield et al. / Aquaculture 302 (2010) 118

D.L. Merrield et al. / Aquaculture 302 (2010) 118

D.L. Merrield et al. / Aquaculture 302 (2010) 118

Oncorhynchus MOS MOS mykiss MOS MOS

D.L. Merrield et al. / Aquaculture 302 (2010) 118

D.L. Merrield et al. / Aquaculture 302 (2010) 118

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