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September | October 2013 EXPERT TOPIC - SALMON
International Aquafeed is published six times a year by Perendale Publishers Ltd of the United Kingdom. All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2013 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058
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36 | InternatIOnal AquAFeed | September-October 2013
According to statistics from the Global Salmon Initiative (GSI) approximately 60 percent of the world’s salmon is farmed. Figures show that in 2011, wild-caught salmon reached approximately 930,000 tonnes. A drop in the ocean compared to the 1,600,000 tonnes produced by aquaculture. Salmon belong to a family of fish known as Salmonidae and based on their distribution, are further classified in to two main genera; Atlantic dwellers (Salmo) and Pacific Ocean based species (Oncorhynchus). The distinguishing factor in this classification is that unlike the Salmo genus, species belonging to the Oncorhynchus genus die after spawning. Due to complex production needs, wide water temperature ranges and biological conditions, farming of Atlantic salmon - the most popular species of Salmonidae - is dominated by just a handful of countries. Currently, the EU, USA and Japan have the largest salmon aquaculture markets.
Atlantic salmon is pisciverous and therefore requires a diet rich in protein and lipids. Farmed fish are usually fed a combination of fish meal and fish oil and although the waste produced from fish processing can be used in certain components of fishmeal production, the risk of transferring disease means it cannot be used directly in fish feed. There is on-going research into supplementing fish feed with plant or microbe-based products, though currently no supplement has been found for pisciverous species (FAO). www.globalsalmoninitiative.org www.fao.org/fishery/en
The Icelandic Ministry of Fisheries and Agriculture states that salmon farming first began in Iceland at the beginning of the 19th century, with the first attempts to rear salmon fry occurring in 1961. The first land-based salmon production
farm was developed in 1978 and by the late 1980s bigger farms were being constructed. Between 1984 and 1987, salmon eggs were imported from Norway. At this time, there were large investments in the production of salmon smolts for export. Later, ocean ranching and cage and landbased farming attracted interest among investors. Ocean ranching involves releasing young reared smolts into rivers and streams. The young smolts use the coastal environment to mature for around a year before returning at which point they are harvested. The country owes much of its farming capabilities to its climate. With unpolluted seas and an abundance of clear water, aquaculture conditions are regarded as among the best in the world in Iceland. In 2007, 600 tonnes of Atlantic salmon were exported. By 2009, there were about 45 registered fish farms on the island. Figures show that of these, about 30 were producing juveniles, mostly for Salmonid on-rearing. According to figures produced
September-October 2013 | InternatIOnal AquAFeed | 37
EXPERT T●PIC by the Directorate of Fisheries, the facilities released around 6 million salmon smolts with an annual return of around 500 metric tonnes. In Iceland, selective breeding and production of healthy Atlantic salmon eggs are supplied on a year round basis. In recent years, major fishery operations have moved into aquaculture, investing in both research and development and the sustainable farming of salmon. www.fisheries.is/aquaculture/ species/atlantic-salmon
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38 | InternatIOnal AquAFeed | September-October 2013
Despite its small size, the Faroese aquaculture industry produces top quality Atlantic salmon. With steady ocean temperatures and strong currents, The Faroe Islands is a prime location for premium salmon production. As a result of this, wild Atlantic salmon from all over northern Europe make their way north of the Faroe Islands to feed. Fish farming in the Faroe Islands began in the 1970s. Nowadays, Faroese fish farming today, which takes place in oceanbased fjords, consists primarily of Atlantic salmon and large rainbow trout and has become a significant component of Faroese economic activity over the past two decades (NASCO). http://salmon-from-the-faroeislands.com
Scotland, which alongside Norway pioneered salmon farming in the 1960s, is now second only to Norway in European production. As smolts are not indigenous to the Shetland Islands, they were originally imported from Norway to establish farms. Atlantic salmon aquaculture in Scotland has increased from 14 tonnes in 1971 to 154,164 tonnes in 2010 and Scotland is now the largest farmed salmon producing country in the EU. It is estimated that the worldwide retail value of Scottish farmed salmon is over GB£1billion. The USA is the largest export market for Scottish farmed salmon, closely followed by France. www.scottishsalmon.co.uk
The development of commercial aquaculture in Norway began in 1970. At present, intensive farming of Atlantic salmon in the country is substantial, accounting for more than 80 percent of total Norwegian aquaculture production (FAO). According to the international environmental organisation, The Bellona Foundation, an average of 20 percent of the oil content in fish feed in Norway comes from vegetable oils. www.fhl.no/english/norwegianseafood-federation-article15-14. html
e APP her re
Salmon farming commenced in Tasmania in the mid-1980s after a report to the Tasmanian Fisheries Development Authority concluded that a salmon farming industry could be successfully developed on the island state. As a result, in 1984 fertilised Atlantic salmon eggs were purchased from the Gaden Trout Hatchery, Jindabyne, New South Wales, Australia from stock originally imported in the 1960s from Nova Scotia, Canada. A sea farm was then established at Dover in the south of Tasmania and a hatchery developed at Wayatinah in the central highlands. The first 53 tonne commercial harvest of Atlantic salmon occurred between 1986 -1987. Nowadays, the Tasmanian industry now produces almost 40,000 tonnes per annum. Nearly 93 percent of Tasmanian salmonid production was sold in the domestic market in 2006. (DPIW) www.tsga.com.au
New Zealand King Salmon's plans for marine farms in Marlborough Sound, New Zealand, may get the go ahead after the High Court dismissed an appeal against them.
The decision of the Board of Inquiry, reached in February 2011, to approve four new salmon farming sites in the Marlborough Sounds was appealed by two parties and that appeal was heard at the High Court in Blenheim in May. The news has been welcomed by the government, “The impacts of these new marine farms on the important recreation and conservation values of the Marlborough Sounds are small. This is about use of only six hectares of more than 100,000 hectares of water space in the Sounds,” said Conservation Minister Dr Nick Smith. “We are a Bluegreen Government that wants jobs and development but also wants to ensure we look after our environment and great kiwi lifestyle. This decision confirms this balanced approach.” “Primary industries are vital for economic growth in our regions, and aquaculture plays an important role in the Marlborough economy. I welcome the news that extra jobs will be created as a result of these new farms,” said Primary Industries Minister Nathan Guy. “This decision is another step forward for New Zealand King Salmon in its plans to establish four new farms, delivering an additional $60 million a year in export income and providing 200 new jobs.”
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September-October 2013 | InternatIOnal AquAFeed | 39
Global salmon farming industry joins forces with new sustainability initiative
by Alice Neal, Associate editor, International Aquafeed magazine
he Global Salmon Initiative (GSI) unites 15 global farmed salmon producers committed to greater industry cooperation and transparency, in order to achieve significant and continuous progress in industry sustainability.
Together, these 15 companies represent 70 percent the global salmon industry, mean-
ing the initiative could have a real impact on salmon aquaculture. The major salmon producing countries of Chile, Norway, Scotland, the Faroe Islands and Canada are all represented in the GSI. Ygnve Myhre, CEO, SalMar, Norway and GSI member, said, “While we have been making attempts at sustainability, salmon farming is a young industry and we recognise that more needs to be done and we can do better. “We know it will take time and will be a continuous process, but through the GSI we have committed to the significant improvement that is needed. This initiative is about significant improvement in sustainability. It is not about satisfaction with the status quo.” The GSI will achieve its aim through global collaboration and research, pooling of resources and sharing knowledge. “What is different is that as the GSI, the companies have committed to helping each other towards improved sustainability. It’s about cooperation, not competition,” said Myhre. Alfonso Marquéz de la Plata, chair of the GSI standards committee and CEO, Empresas AquaChile S.A., Chile, said, “We cannot choose between a healthy environment and healthy food, we need both. This initiative is a practical approach to achieving both. While meeting the standard at a global level will be a significant challenge, this is a major commitment from the salmon farming industry and we hope that through GSI collaboration, we can get there together.” The initial impetus for the GSI came from a meeting in 2011 which was attended by a number of CEOs. At that meeting, the CEOs heard about significant progress other industries had made in sustainability by working together. That group of CEOs decided to meet again and invite other CEOs and in due course it was agreed to form the GSI. Currently, the GSI is focusing on biosecu-
Chris Ninnes, chief executive, ASC, “GSI’s commitment to significantly improving the sustainability of salmon farming mirrors ASC’s aim to transform aquaculture towards environmental sustainability and social responsibility"
Ygnve Myhre, CEO, SalMar, Norway and GSI member, “While we have been making attempts at sustainability, salmon farming is a young industry and we recognise that more needs to be done and we can do better"
rity, feed and nutrition and meeting industry standards. In terms of feed ingredients, the GSI is keen to find sources that do not put further stress on marine resources. The GSI is considering utilizing by-products and is working closely with the FAO to assess availability of these resources. The GSI has chosen the Aquaculture Stewarship Council (ASC) as its accreditation body and aims to have all its members meet the ASC Salmon Standard by 2020. Chris Ninnes, chief executive, ASC said, “GSI’s commitment to significantly improving the sustainability of salmon farming mirrors ASC’s aim to transform aquaculture towards environmental sustainability and social responsibility. “A commitment at this scale presents an unprecedented opportunity to realise a meaningful reduction in the environmental and social impact of the sector. It is a huge statement of leadership intent to tackle these issues.” The initiative ties in with ASC plans to launch a certified salmon to the market in early 2014. “I consider it extremely positive that a major proportion of the salmon farming industry is voluntarily seeking to become environmentally responsible and to do this in a transparent way so that all can see the reduction of industry impact. “Transparency is one of the cornerstones of ASC. The standards require an unprecedented amount of public disclosure of farm-level data from certified farms that are currently not publicly available in most cases. GSI members are aware of these requirements. However, as an industry-led initiative and by working together members are well placed to meet them as they achieve certification.” The initiative has been warmly welcomed by the aquaculture industry. Mary Ellen Walling, executive director, British Columbia Salmon Farmers’ Association, Canada said, “This initiative recognises that there is no limit to how sustainable you can be. You don’t reach the highest level and stop; there is always room for improvement, always more you can learn. This collaboration will benefit the industry in BC and around the world.” GSI member companies include Acuinova Chile; Bakkafrost; Blumar; Cermaq; Compañía Pesquera Camanchaca; Empresas AquaChile; Grieg Seafood; Lerøy Seafood Group; Los Fiordos; Marine Harvest; Norway Royal Salmon; SalMar; Multiexport Foods SA; The Scottish Salmon Company; Scottish Sea Farms. More InforMatIon:
40 | InternatIOnal AquAFeed | September-October 2013
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and outcompete endogenous bacteria and pathogens. Investigating these topics effectively in in vivo models can be difficult as they are time consuming and costly. Furthermore, as the EU has recommend reductions of in vivo experiments and the numbers of animals used in experiments (Revision of the EU directive for the protection of animals used for scientific purposes [Directive 86/609/EEC]; 8th of September 2010), attempts have been made to use alternative ex vivo methods (e.g. the Ussing chamber, everted sack and intestinal sack methods). The first aim of the present study was to investigate possible effects of a prebiotic feed on epithelial histology and indigenous GI tract microbiota in the proximal intestine (PI) and distal intestine (DI) of Atlantic salmon. Furthermore, the same effects, including morphological changes of epithelial cells after ex vivo exposure of the intestinal tract to Carnobacterium divergens, a probiotic bacterium, are investigated by light microscopy and electron microscopy. The result of Carnobacterium exposure is of high importance to evaluate as translocation and cell damage are negative criteria when evaluating the use of probiotics in endothermic animals as well as in fish. The second aim of the present study was to evaluate the bacterial community of the PI and DI of salmon fed control or prebiotic diets, before and after ex vivo exposure to probiotic bacteria, in order to investigate if the indigenous GI tract microbiota is modulated by the different treatments. Finally, we addressed the issue as to whether carnobacteria isolated in the ex vivo studies were able to inhibit in vitro growth of the pathogenic bacteria Yersinia rückeri and Aeromonas salmonicida ssp. salmonicida.
table 1: Dietary formulation and chemical composition of the experimental diets % Fishmeal north atlantic fish oil Vegetable protein concentrates1 Vegetable oil Carbohydrate-based binders2 Micro premixes3 Chemical composition (%) 31.25 13.50 25.76 14.01 13.00 2.48
Evaluation of prebiotic and probiotic effects on the intestinal gut microbiota and histology of Atlantic salmon
by Mads Kristiansen, Einar Ringø Norwegian College of Fishery, Faculty of Bioscience, Fisheries and Economics, University of Tomso, NorwayAquamedical Contract Research, Vikan, Norway; Daniel Merrifield, Aquaculture and Fish Nutrition Research Group, School of Biomedical and Biological Sciences, University of Plymouth, UK; Jose Gonzalez Vecino, EWOS Innovation AD, Dirdal, Norway and Reidar Myklebust, Molecular Imaging Center, Institute of Biomedicine, University of Bergen, Norway
Moisture Protein4 Fat4 nFe4 ash4
1 Incudes soy protein concentrate, pea
6.9 44.2 29.1 1.6 8.4
protein concentrate, wheat gluten, sun flower meal. 2 Includes wheat and pea starch 3 Includes vitamin, mineral, amino acid and pigment premixes and 0.2% eWoS prebiosal® added to the prebiotic diet (at the expense of an equal volume of carbohydrate-based binders) 4 dry weight basis
day with duration of 2.5 hour between each feeding for a period of 15 weeks. During the feeding period the water temperature and salinity ranged, with season, from 5.3-12.9 °C and 26.7-30.9 gl-1. The samplings were carried out at two different points: at the start (week 0) and at the end of the trial (week 15). An overview of the different treatments and groups is listed in Table 2.
oday it is generally accepted that the three major routes of infection in fish are through: a) skin, b) gills and c) the gastrointestinal (GI) tract. The GI microbiota, including lactic acid bacteria (LAB), have been suggested to be important in fish health and it has been suggested that the autochthonous gut bacterial community may be responsible for controlling the colonization of potential pathogens by adhesion competition and production of antagonistic compounds. If the GI tract is involved as an infection route, scientists have address whether probiotic bacteria are able to adhere to and colonise mucosal surfaces
The probiotic bacterium used in this Two hundred and forty vaccinated Atlantic experiment was Carnobacteriumdivergens salmon (Salmo salar L.) were held at the strain Lab01 originally isolated from juvenile EWOS Innovation AS Research Station, Atlantic salmon fed a commercial diet. The Dirdal, Norway. The average weight at the bacteria were stored in glycerol-containing start of the experiment was 350 g. Two hun- cryotubes at -80 °C and inoculated into tryptic dred and forty fish were distributed equally soy broth (Difco, USA) with glucose (10 g l-1) (i.e. 40 fish per tank) into six tanks supplied with 500 litres of seawater and Table 2: Experimental treatments applied to Atlantic salmon two diets were offered (i.e. triplicate intestine fed control and prebiotic diets tanks per diet). The control diet and treatment type of type of Week of prebiotic diet had the same ingredient grouop treatment feed feeding composition (Table 1) and differed only in the inclusion of 0.2 percent EWOS 0 1 Saline Control prebiosal® in the prebiotic diet. EWOS 1 prebiosal®, is described as a multi-com0 Control 2 C. divergens ponent prebiotic specifically designed for 15 3 Saline Control salmonid fish; more detailed information 15 4 C. divergens2 Control about the composition of EWOS prebi15 5 Saline Prebiotic osal® is not available for commercial 15 6 C. divergens2 Prebiotic reasons. Feeding was conducted twice a
42 | InternatIOnal AquAFeed | September-October 2013
Table 3: Cultruable heterotrophic bacterial levels (log CFU g-1 wet weight) and identity (as determinedfrom phenotypic characteristics and 16S rRNA sequence analysus) obtained from different groups after the ex vivo assay
Proximal intestine Group tVC (log no CFU g-1) Bacteria % tVC (log CFU g-1) no
Distal intestine Bacteria %
Psychrobacter aquimaris - 16.7% Psychrobacter glancincola - 16.7% Psychrobacter spp - 66.6% Carnobacterium divergens - 100% Carnobacterium divergens - 70.6% Pseudomonas fluva - 17.6% Pantoea spp - 5.9% Gammaproteobacteria - 5.9% Carnobacterium divergens - 87.5% Pseudomonas spp - 12.5% Carnobacterium divergens - 29.8% Carnobacterium spp - 51% Pseudomonas antartica - 2.1% Pseudomonas korensis - 2.1% Enterbacter hormaechi - 8.5 Gammaproteobacteria - 4.3% Uncultured bacterial clone CK20 - 2.1% Psychrobacter marincola - 4% Pseudomonas sp - 8% Carnobacterium divergens - 20% Carnobacterium spp - 68%
Psychrobacter glancincola - 9.0% Psychrobacter spp - 36.3% Pseudoalteromonas - 36.3% Brevibacterium sp. - 9.0% Moraxella sp. - 9.0% Carnobacterium divergens - 100% Carnobacterium divergens - 33.3% Pseudomonas fluva - 6.6% Shewanella baltica - 6.6% Vibrio splendidus - 13.3% Gammaproteobacteria - 40% Carnobacterium divergens - 100%
Carnobacterium divergens - 25% Carnobacterium spp - 52.3% Pantoea spp. - 18.2% enterobacter spp. - 4.5%
acinetobacter sp. - 5.6% Carnobacterium divergens - 94.2%
*N = number of isolates identified
and NaCl (10 g l-1), viz. TSBgs medium. After approximately 24 hours of preinoculation at room temperature with an agitation of 190 rpm, 1 percent of the preculture was transferred to new TSBgs medium and growth (same growth conditions as above) was measured by optical density for evaluation of the growth cycle (data not shown). Bacterial viability was confirmed by plating bacterial suspensions on tryptic soy agar (Difco) + glucose (15 g l-1) and NaCl (15 g l-1) (TSAgs) plates. The results obtained from this study were used to calculate the bacterial concentration in the experimental bacterial solutions.
September-October 2013 | InternatIOnal AquAFeed | 43
EXPERT T●PIC the protocol from a commercial kit (DNeasy Blood and Tissue, Qiagen, USA). Specific treatment for Gram-positive and Gramnegative isolates was carried out according to the manufacturer’s instructions. TemplateDNA was diluted to a concentration of approximately 20-30 ng μl-1 using Milli-Q water. The PCR mix constituted of 8 μl of template-DNA, 36 μl Milli-Q water, 5 μl 10x buffer F511, 0.25 μl dNTP, 0.25 μl27F forward primer, 0.25 μl 1492R reverse primer and 0.25 μl DNA-polymerase yielding a total volume of 50μl. PCR thermal cycling consisted of initial denaturation of 94 ºC, followed by 35 cycles of 94 ºC for 20 s, 53 ºC for 20 s and 72 ºC for 90 s with a final extension step of 72 ºC for 7 min. To verify PCR products, samples were run on gel electrophoresis. The PCR-products were desalted by mixing 20 μl of PCR-product with 40 μl of 100 percent ethanol and 2 μl of 3M NaOAc (pH 5.3) and vortexed well. Samples were then incubated on ice for 30 min followed by centrifugation for 20 minutes at 14,000 gusing an Eppendorf Microcentrifuge Model 5417R. The supernatant was removed and pellet washed in 100 μl of 80 percent ethanol and centrifuged for another 5 minutes at 14,000 g. The supernatant was removed and the pellet dried at room temperature for 60 minutes. The pellet was then resuspended in 30 μl of Milli-Q water. Purified PCR products were sequenced as described elsewhere. The resultant nucleotide sequences were submitted to a BLAST search in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to retrieve the closest known alignment identities for the partial 16S rRNA sequences. Gene sequences that showed higher than 95 percent similarity to a genus or species in GenBank were categorized accordingly. In vitro growth inhibition of pathogens by LAB isolated form the ex vivo studies Eleven randomly chosen LAB isolated from the intestinal tract after ex vivo exposure and one type strain, Carnobacterium inhibens (CCUG 31728), were tested for antagonistic effects against two different fish pathogens. The pathogenic bacteria used in the present investigation were Yersinia rückeri (CCUG 14190) and Aeromonas salmonicida ssp. salmonicida (Ass 4017). C. inhibens (CCUG 31728) was used as a positive control as previous investigations have demonstrated that this strain has an inhibiting effect towards V. anguillarum and A. salmonicida. In vitro growth inhibition of the two fish pathogens by the twelve LAB was tested using a microtitre plate assay described in detail by Ringø and co-authors. This method has been used in two recent studies. The pathogenic bacterial levels at the start of assays were 106 cells ml-1. Positive in vitro growth inhibition was defined when no growth (turbidity < 0.05 at optical density; OD600 nm) of the pathogen
Ex vivo exposure to bacteria
Three fish were randomly selected from two of the tanks fed each diet and killed with a blow to the head. The entire intestine, from the last pyloric caeca to the anus, was removed aseptically and intestinal content was gently squeezed out, before the intestine was flushed three times with sterile saline solution (0.9% NaCl), in order to remove the allochthonous gut bacteria. The posterior end was tightly tied with cotton thread before filling (ca. 1.5 ml) with the appropriate assay solution (Table 2), tying the anterior end and suspending the sealed intestinal tube in sterile saline solution. The intestinal sacks were then incubated at 10 ºC for one hour. After incubation the intestine was cut open, the contents discarded and flushed three times with sterile saline solution.
Post ex vivo bacterial assays
Samples for bacteriology from each segment from the first sampling point (groups 1 and 2) were prepared by homogenizing 1 g of
intestinal tissue (PI or DI) in 1 ml sterile saline using a Stomacher (Seaward Laboratory, UK). Gut samples for bacteriology from the second sampling (groups 3-6) were prepared by gently scraping off mucus with a sterile scalpel. Thereafter, the segments were weighed. Both the homogenates and mucus were used to create serial ten-fold dilutions which were spread plated (100 μl) on TSAgs plates and incubated at 6 ºC for up to 1 week to determine viable counts of culturable heterotrophic bacteria. After sub-culturing on TSAgs to achieve pure cultures, phenotypic bacterial identification (Gram stain, colony morphology, oxidase - and catalase tests and glucose fermentation) was carried out on random colonies from all plates containing between 10-300 colonies. A total of 168 bacterial strains were isolated from the two sampling points.
16S rRNA characterization of isolates
The bacterial DNA was isolated following
44 | InternatIOnal AquAFeed | September-October 2013
EXPERT T●PIC was detected. Sterile growth media and the pathogens were used as controls. Growth (at OD600) of the pathogens without addition of sterile supernatant of LAB was approximately 0.6. Measurements were carried out each hour using an automatic plate reader (Bioscreen C, Labsystems, Finland).
Table 4: Identification of LAB strains and pathogen antagonistic activity of extracellular products used in the in vitropathogen asays
Isolate code 33 40 75 84 14 57 17 154
Source group Group 2 Group 2 Group 3 Group3 Group5 Group5 Group 5 Group 5
Intestinal region Proximal Distal Proximal Distal Proximal Proximal Distal Distal
Closest known species C. divergens C. divergens C. divergens C. divergens Carnobacterium sp C. divergens Carnobacterium sp C. divergens
Identity (%) 98 98 99 100 86 99 99 92
lHICa_53_4 lHICa_53_4 lHICa_53_4 lHICa_53_4 H126a lHICa_53_4 H126a lHICa_53_4
FJ656716.1 FJ656716.1 FJ656716.1 FJ656716.1 eF204312.1 FJ656716.1 eF204312.1 FJ656716.1
Y. ruckeri + + + + + + +
a. Salmon + -
Samples for light C. divergens 173 Group 4 Proximal lHICa_53_4 FJ656716.1 99 + microscopy (LM) and C. divergens 127 Group 4 Distal lHICa_53_4 FJ656716.1 99 + transmission electron C. divergens 99 Group 8 Proximal lHICa_53_4 FJ656716.1 99 + microscopy (TEM) were collected by excising *_originally isolated from the digestive tract of Atlantic salmon (salmo salar)  approximately 5 mm from the posterior part of the PI and DI. The samples were immedi- divergens (group 4). In both dietary groups, munity which consisted of 4 different bacterial ately fixed in McDowells fixative and stored a similar bacterial level (~log 6.70 CFU g-1) genera. Of these, ten strains were indentified at 4 ºC until processing. TEM and LM sam- was detected in DI exposed to C. divergens. to genus level and one strain was identified ples were processed as described elsewhere However, a higher bacterial level (log 2.69 to species level. The bacteria identified to Morphological observations were made from CFU g-1) was observed in the DI of control genus level belonged to Pseudoalteromonas, multiple micrographs (8) from each intestinal fed fish exposed to saline than that of prebi- Psychrobacter, Moraxella and Brevibacterium, while the last strains showed high similarity region from two fish within each group. otic fed fish (log 1.71 CFU g-1). The following morphological parameters were Isolation and identification of bacteria after (98 percent) to Psychrobacter glacincola. Microbiota of fish fed control diet and observed; detached microvilli, enterocytes ex vivo exposure detached from the basal membrane, disinteA total of 168 bacterial strains were intestines exposed to C. divergens (group 2): grated cell junctions, presence of goblet cells, isolated from the two samplings. Among All bacteria isolated from PI and DI of fish presence of absorptive vacuoles and presence these, 40 isolates were isolated from the first exposed to C. divergens at the first sampling of intraepithelial lymphocytes. sampling point and 128 isolates were isolated (group 2) were identified as C. divergens. This from the second sampling point. All isolates observation indicates that C. divergens are able were tested for morphology and biochemical to adhere to the intestinal mucosa in both Results properties (colony morphology, Gram-testing, segments. Bacterial levels after ex vivo exposure The adherent bacterial levels, as deter- oxidase - and catalase tests and glucose mined by using a stomacher (groups 1 and 2) fermentation). Week 15 One hundred and eleven isolates were or by the collection of mucus (and subsequent Microbiota of fish fed control diet and weighing of the segments the mucus was further identified by partial sequencing of the intestines exposed to saline (group 3): After 15 removed from) (groups 3-6), did not seem to 16S rRNA gene. Isolates not identified by 16S weeks of feeding on the control diet, the isodiffer which indicates that the different sam- rRNA gene sequencing but showing similar lated strains (17) from the PI exposed to saline pling methods were similarly effective. Table biochemical and physiological properties to were dominated by C. divergens; 70.6 percent 3 presents an overview of the autochthonous those isolates identified by 16S rRNA genes of the isolates were identified as C. divergens, bacterial levels isolated from each segment were defined as ‘-like’. Table 3 provides an 17.6 % were identified as Pseudomonas fulva, and each group exposed to either saline or C. overview of the different bacterial species 5.9 % belonged to Pantoea spp. while 5.9 % of divergens. All values are expressed as log col- isolated in each experimental group. the isolates were identified as members of the ony forming units (CFU) g-1. Autochthonous class Gammaproteobacteria. The bacteria isolated from the DI were identified as C. divergens, bacteria isolated from intestines of fish fed Week 0 the control diet at the experimental start and Microbiota of fish fed control diet and two strains as Vibrio splendidus, one strain as exposed to saline was approximately log 1.7 intestines exposed to saline (group 1): Shewanella baltica, one strain as Pseudomonas CFU g-1 in both PI and DI, while the number Analysis of the adherent microbiota in the fulva and six other strains were identified as of bacteria isolated from intestines of fish PI of fish fed the control diet and exposed Gammaproteobacteria. Microbiota of fish fed control diet and exposed to C. divergens was log 6.04 CFU g-1 to sterile saline (group 1) revealed that all isolates belonged to the genus Psychrobacter. intestines exposed to C. divergens (group 4). In in PI and log 5.56 CFU g-1 in DI. After 15 weeks of feeding slightly higher Of the 12 strains isolated from the PI of the intestine of fish fed the control diet for 15 values were present in PI of fish fed the this group, two strains showed 96 percent weeks and exposed to C. divergens, the identiprebiotic diet post exposure to saline or C. similarity to Psychrobacter aquimaris, two fied bacterial strains isolated from both PI and divergens compared to fish fed the control strains were identified as Psychrobacter gla- DI were dominated by C. divergens. Only one diet. Indeed, the bacterial level in the prebi- cincola while eight strains were identified as strain, identified as Pseudomonas spp., isolated from the PI of 1 fish did not belong to the otic fed fish intestine exposed to C. divergens Psychrobacter spp.- like. The DI of fish exposed to saline at the first species C. divergens. (group 6) was 234 percent greater than that Microbiota of fish fed prebiotic diet and of the control fed fish intestine exposed to C. sampling point showed a more diverse comSeptember-October 2013 | InternatIOnal AquAFeed | 45
EXPERT T●PIC intestines exposed to saline (group 5): The intestine exposed to saline of fish fed the prebiotic diet for 15 weeks showed higher diversity compared to the other groups exposed to saline (groups 1 and 3). Of the 47 strains isolated from the PI, 14 were identified as C. divergens, one as Pseudomonasantarctica, one as Pseudomonas koreensis, four as Enterobacter hormaechei and one as uncultured bacterial clone CK20. The remaining isolated strains were identified as members of the Carnobacterium and Acinetobacter genera. The dominant bacteria in the PI of this group belonged to carnobacteria (81%) and 30 percent of total isolates were identified as C. divergens. The bacterial composition of the isolates from the DI of fish fed the prebiotic diet for 15 weeks were relatively low in diversity. Of the total number of strains isolated (44) from the DI exposed to saline 34 were identified as Carnobacterium, eight strains showed high similarity (%) to Pantoea spp. and two strains belonged to the genus Enterobacter. Microbiota of fish fed prebiotic diet and intestines exposed to C. divergens (group 6): In group 6, fish fed the prebiotic diets for 15 weeks and exposed to C. divergens, the isolated strains in the PI were dominated by C. divergens and C. divergens-like strains. Of the 22 carnobacteria isolated, five were identified as C. divergens by 16S rRNA sequencing while 17 isolates were identified as C. divergens-like. Three other isolates were identified as members of the genera Pseudomonas (2 strains) and Psychrobacter (one strain). Of the 17 strains isolated and idesntified from the DI of group 6, C. divergens and C. divergens-like strains dominated with only one isolate, which showed high similarity (99 percent) to Acinetobacter spp., not belonging to this species.
Light microscopy (LM): All LM micrographs, both from PI and DI of the prebiotic groups (5 and 6) showed no morphological differences compared to the control feeding regime (groups 1-4). All intestinal sections examined appeared normal and healthy; no signs of detached enterocytes, necrotic enterocytes, widened lamina propria or necrosis were observed and the number of goblet cells were similar in both treatments (examples are displayed in Figure 1). Transmission electron microscopy (TEM): Similar to the observations using LM, TEM revealed no differences between treatments or exposure groups; all micrographs revealed healthy epithelial brushborder, no deteriation of tight junctions was observed and microvilli appeared uniform. The presences of rodletlike cells (as shown in Figure 2) were present FIAAPisland:Layout 1 30/8/13 14:26 Page 1 in the PI and DI of all groups. The numbers of rodlet cells present in the PI displayed great differences between individual fish but were always observed in the upper half of the epithelium, above the underlying intraepithelial lymphocytes. In vitro growth inhibition of two fish pathogens by extracellular extracts of LAB isolated from ex vivo studies 8 – 10 April 2014 . Bangkok International Trade & Exhibition Centre (BITEC), Bangkok, Thailand Identification by partial sequencing of the 16S rRNA genes of the eleven LAB strains isolated from the ex vivo experiments and subsequently used in the in vitro pathogen antagonism assays are displayed in Table 4. The results show that growth inhibition of Y. rückeri was obtained from extracellular extracts from all strains of carnobacteria isolated from the ex vivo experiment. However, in vitro growth inhibition of A. salmonicida ssp. salmonicida was only obtained from the extracellular extract of C divergens isolate 57. The extracellular products from the positive control, C. inhibens CCUG 31728, did not inhibit the growth of A. salmonicida ssp. salmonicida. FIAAP Asia 2014 is the only dedicated trade show and conference organised specifically for feed ingredients,
Asia’s foremost exhibition and conferences for the ingredients and additives used in the production of animal feeds, aquafeeds and petfoods
additives and formulation within the dynamic and growing region of South and South East Asia.
New for 2014 Now including the first ASEAN Feed Summit Specialist conferences The exhibition will be supported by its own specialist conferences. They will include: The FIAAP Conference 2014 Petfood Forum Asia 2014 Aquafeed Horizons Asia 2014 The Thai Feed Conference 2014 Supported by The Thailand Convention and Exhibition Bureau Co-located with VICTAM Asia 2014 www.victam.com Contact details For visitor, exhibition stand space and conference information please visit: www.fiaap.com
The ex vivo intestinal sack method has been used in several studies to evaluate possible histological changes in the fish intestine after exposure to high levels of LAB. The result of LAB exposure to the intestine is of high importance as translocation and cell damage have been proposed as important criteria when evaluating
46 | InternatIOnal AquAFeed | September-October 2013
EXPERT T●PIC the use of probiotics in endothermic animals spp. are commonly reported in the GI tract of bacteria have previously been isolated from as well as in fish. Recently, the effect of ex vivo fish, and these bacteria have previously been the intestine of Atlantic salmon. The bacteLAB exposure on the gut microbiota in fish isolated and identified from the GI tract of sal- rial composition of the PI observed in the was documented, but to the authors’ knowl- monids. Carnobacterium spp. have often been prebiotic fed fish exposed to saline (group edge the effect of prebiotic supplementation reported to be components of the gut micro- 5) showed greater bacterial diversity than and ex vivo LAB exposure of the fish intestine biota of salmonids; indeed, C. maltaromaticum, that observed from control fed fish exposed C. mobile, C. divergens and Carnobacterium spp. to saline (groups 1 and 3). The majority of has not been investigated. The culturable bacterial levels recovered have been identified from Atlantic salmon. the bacteria from the PI of group 5 were on TSAgs plates from groups exposed to The consistency of isolation of these species C. divergens and Carnobacterium spp., which saline were relatively low, ranging from log indicates that these might be common core together accounted for 81% of all the isolated 1.72 to 2.34 CFU g-1. These values are low components of the GI microbiome of Atlantic bacteria. The two other bacterial species that compared to autochthonous levels previously salmon and are likely to be of importance to were isolated from this group were Pantoea spp. and an unidentified member of the reported in Atlantic salmon and rainbow trout the host. The bacterial composition isolated from class Gammaproteobacteria. The abundance of Oncorhynchus mykiss. This is likely due to the thorough rinsing process, three times prior the DI in the first control group exposed culturable adherent Carnobacterium spp. (77% and three times post probiotic/saline expo- to saline (group 1) were dominated by of isolates identified) was higher in the DI of sure. Culturable adherent bacteria in the PI Pseudoalteromonas spp. and Psychrobacter spp., the prebiotic fed fish (group 5), compared to observed in the prebiotic group exposed to while in the other control group (group 3) control groups (group 1 = 0% and group 3 saline (group 5) was log 2.34 CFU g-1. The Acinetobacter spp. and C. divergens were the = 36%). These results suggest that the prebifact that the value from this group is higher dominant bacteria isolated. All of these listed otic supplementation elevates autochthonous VICTAMisland:Layout 1 30/8/13 14:22 Page 1 than in control groups exposed to saline water (log 1.72 and 2.08 CFU g-1), might be due to a feeding effect of the prebiotic diet. However, this hypothesis merits further investigations. Investigations of the qualitative and quantitative bacterial composition of the intestinal microbiota were based on the study of 168 pure cultured bacterial isolates. These isolates were biochemi8 – 10 April 2014 . Bangkok International Trade & Exhibition Centre (BITEC), Bangkok, Thailand cally tested in order to obtain a general classification. From this classification 111 isolates were selected by the lottery method and identified by 16S rRNA gene sequencing analysis. The bacterial levels observed in groups exposed to saline varied between segments and feeding regime. By comparing different feeding groups it is clear that the indigenous microbiota of the PI were affected by the diet, while the effect of prebiotic feeding on the microbiota was less clear in the DI. In the intestine exposed to C. divergens the average number of bacteria were higher in the PI when the fish were fed the prebiREAL BRE WER otic diet compared to fish fed S’ YEAS the control diet. Whether these VICTAM Asia 2014 is the largest trade show within South and South East Asia for displaying the T latest “ Made in equipment and technology used in the production of animal feeds, aquafeeds and dry G petfoods. findings can be related to a higher ermany ” C. divergens colonization success New for 2014 Supported by in prebiotic fed fish merits further Now including the first The Thailand Convention ForFeed Leiber`s ASEAN Summit specialty yeast products, and Exhibition Bureau investigations. “Made in Germany” is a seal of quality. The bacterial composition Specialist conferences Co-located with The exhibition will be supported FIAAP Asia 2014 and from the PI in control groups, i.e. by its own specialist conferences: GRAPAS Asia 2014 Multibiotic effect of Leiber yeast - vitality, health and performance fish fed control diet, and thereafwww.fiaap.com / www.grapas.eu for fish. The FIAAP Conference 2014 Petfood Forum Asia 2014 ter exposed to saline were domiContact details Aquafeed Horizons Asia 2014 For visitor, exhibition stand space and nated by members of a few genThe Thai Feed Conference 2014 www.leibergmbh.de conference information please visit: era (Psychrobacter, Carnobacterium www.victam.com Biomass Pelleting Asia 2014 and Pseudomonas). Psychrobacter, Leiber GmbH · Hafenstraße 24, 49565 Bramsche, Germany · Tel +49 (0) 5461 9303-0 · Fax +49 (0) 5461 9303-28 · www.leibergmbh.de · email@example.com Carnobacterium and Pseudomonas
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September-October 2013 | InternatIOnal AquAFeed | 47
EXPERT T●PIC that enterocytes showed no signs of junctional rupture from the basement membrane which is in contrast to observations of the PI of Atlantic salmon after exposure to Vibrio anguillarum and A. salmonicida. TEM observations confirmed the findings observed in LM regarding a lack of histological changes. TEM revealed no observable differences between the groups in respect to the presence of cell debris in the lumen, the amount of mucus, the number bacteria – like particles in the lumen and between the microvilli, disorganized microvilli and disintegrated tight junctions. The enterocytes within all groups displayed normal cell contacts with unaffected tight junctions and zona adherens. The fact that C. divergens did not inflict damage to the intercellular unction is great importantance since the loosening of these junctions contributes to a paracellular port of entry for potential pathogens. Rodlet cells were present in "The histological effect of exposing the large numbers in the PI of all groups, while in the DI the number GI tract of Atlantic salmon to high levels observed were lower. Since groups of the C. divergens was investigated exposed to C. divergens did not display any clear differences in number by light and electron microscopy" of rodlet cells compared to groups exposed to saline, their presence ex vivo studies the same C. divergens strain in those groups may therefore not be related was identified to dominate both the PI and to an immunological function towards the DI after exposure. C. divergens levels were in exposed bacteria. On the other hand, the the range of 104-106 CFU g-1 intestine which role of rodlet cells as immune cells and their indicates that the bacteria are able to populate large number in the PI compared to the DI and potentially colonize the intestinal mucus may be a defense function towards potential and out-compete other adherent bacteria invading bacteria of the PI. Since the PI has after only one hour of exposure. These results been confirmed as being an infection route are in accordance with corresponding studies for pathogenic bacteria by several studies, the in that LAB are able to colonize the intestine role of rodlet cells as immune cells in the PI is possible and warrants further investigation. of Atlantic salmon after one hour exposure. The antimicrobial effects of LAB have long Despite the plethora of information available on the prebiotic efficacy of elevating pro- been utilized in food preservation by fermentabiotic colonization (i.e. synbiotics) in various tion and several comprehensive reviews have terrestrial species, little information is available been published on the ability of LAB to proin fish. Further studies should focus on this duce proteinaceous antimicrobial substances. In topic as the present study demonstrated that fish studies, the antagonistic effect of LAB has the presence of the dietary prebiotic, prebi- been carried out on Gram-negative fish pathoosal®, elevated the proportion of carnobac- gens such as V anguillarum and A salmonicida. teria from 71 - 81 percent in the DI (as well In the present study strong growth inhibition as elevating total bacterial levels, effectively of Y. rückeri was recorded from extracellular quadrupling the number of carnobacteria) and extracts from late exponential growth phase from 33% to 77% in the PI (although the total from all of the eleven Carnobacteria strains isolated from the ex vivo experiments. However, bacterial population was lower). The histological effect of exposing the GI the ability of the isolated strains to inhibit tract of Atlantic salmon to high levels of the growth of A. salmonicida ssp. salmonicida was C. divergens was investigated by light and elec- only observed from one strain isolated from tron microscopy. Furthermore, the intestinal the PI. The fact that only one (isolate 57) of the effects of feeding a prebiotic diet to Atlantic 11 strains displayed inhibitory effects towards A. salmonicida ssp. salmonicidais in accordance salmon were evaluated. Results from LM-investigations in the with the results of Ringø who observed a lack present study showed no apparent his- of antagonism when challenging A. salmonicida topathological changes of the epithelium in ssp. salmonicida to extracellular extracts from the PI or DI, after exposure of C. divergens. C. divergens strain Lab01. These results indicate In particular the micrographs demonstrated that the production of extracellular products Carnobacterium spp. levels, particularly in the DI. To the authors’ knowledge there is very little information regarding the effect of prebiotics on carnobacteria within the GI tract of fish. However, some studies suggest that the carnobacteria populations within the GI tract of salmonids are effected by various dietary factors such as krill meal and oxytetracycline in Atlantic salmon and dietary carbohydrates in Arctic charr (Salvelinus alpinus L.). However, it was observed that the presence of dietary inulin (a prebiotic-type carbohydrate) tended to lower culturable autochthonous carnobacteria levels (by ca. 90%) in the hindgut of Arctic charr and also elevated the proportion of C. maltaromaticum at the expense of C. divergens. These findings suggest that different prebiotics may influence different carnobacteria strains in different fish species. In all groups exposed to C. divergens in the only, might not be sufficient for strains of C. divergens in late exponential growth phase, to inhibit growth of A. salmonicida ssp. salmonicida. The positive control bacteria, C. inhibens which Jöborn et al. reported to display antagonistic effect against A. salmonicida, showed no sign of antagonism in the present study. This observation therefore indicates that antagonisitic activity of C. inhibens is only effective when cells are actively incubated together or that antagonistic extracellular products are only produced by C. inhibens in the presence of A. salmonicida. The ability of C. divergens as useful probiotics with effects against Y. rückeri and A. salmonicida have previously been reported in vivo and in vitro. Kim and Austin observed that dietary provision of C. divergens strain B33, isolated from the intestine of healthy rainbow trout, increased survival of rainbow trout against A. salmonicida and Y. rückeri challenge by 60 percent compared to the control group. Even though strains of C. divergens show antagonistic effects against pathogens, the precise mechanism of action of antimicrobial compounds isolated from fish remains unclear, but suggestions about their ability of penetrating cell walls by forming pores and channels, thus rendering it more fragile and incapable of carrying out normal metabolism has been proposed. In order to confirm the in vitro probiotic effect of C. divergens against Y. rückeri in Atlantic salmon, further investigations should therefore include in vivo challenges studies. By further applying electron microscopy, the physical interference mechanisms between C. divergens and Y. rückeri in the GI tract might be observed.
The authors thank the technical staff at EWOS Innovation AS for feed manufacture, analysis and running the feeding trial and thank Dr. Sigmund Sperstad and Dr. Chun Li, Norwegian College of Fishery Science, University of Tromsø for their inestimable help during 16S rRNA gene sequencing and in vitro growth inhibition. We also thank Randi Olsen and Helga Marie By at the EM department at University of Tromsø, and Anne Nyhaug at Molecular Imaging Centre, Institute for Biomedicine, University of Bergen for their inestimable help during light and electron microscopy analysis. This study was partially supported by grants from the Norwegian MABIT-program (project number AF0038).
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48 | InternatIOnal AquAFeed | September-October 2013
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