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Aquaculture and Fisheries Management 1994, 25 , 623-629

Microfiora of Arctic charr, Salvelinus alpinus (L.) :gastrointestinal microflora of free-living fish and effectof diet and salinity on intestinal microfloraE . R I N G 0 University of Troms0, Institute of Biology an d Geology, Troms0, NorwayE. S T R 0 M Troms0 College of Health Care Education, Troms0, Norway

Abstract. The adheren t aerobic bacterial flora present in the gastrointestinal tract and faeces offree-living A rctic cha rr, Salvelinus alpinus (L. ), from Lake Takvatn, Northern Norway, wereidentified both qualitatively and quantitatively. Approximately 10 bacteria g~' were found inboth the small and large intestines. The predominant bacterial species were identified asAeromonas, Enterobacteriaceae, Micrococcus and Lactobacillus. Other microorganismsisolated included Acinetobacter, Cytophaga, F lavobacterium, Morax ella, Pseudom onas,Vibrio, Coryneforms and Streptococcus. The intestinal microflora of free-living fish wasdominated by Aeromonas and Lactobacillus, but the intestinal bacterial flora of wild fishtransferred to hatchery was affected by feeding them eithe r a capelin roe diet or a commercialfeed in fresh and sea water. Approximately 55% ofthe bacterial flora in intestinal contents fromfish fed the capelin roe diet was Enterobacteriaceae when the fish were held in fresh and seawater. However, when the wild-caught charr were fed a commercial diet in fresh water,Aeromonas and Pseudomonas dominated in faeces, while Vibrio and Pseudomoruis werepredominant in the diet group held in sea water.

IntroductionStudies have been conducted on the bacterial flora associated with the intestinal wall indifferent regions of the alimentary tract of free-living chum salmon, Oncorhynchus keta(Walbaum) (Trust & Sparrow 1974, Trust 1975), brook trout , Salvelinus fontinalis (Mitchill),golden trout, Oncorhynchus aguabonita (Jordan), rainbow trout, Oncorhynchus mykiss(Walbaum) (Trust & Sparrow 1974), and hatchery-cultured rainbow trout (Austin &Al-Zahrani 1988). However, no information exists about the adherent microflora in thegastrointestinal tract of Arctic charr, Salvelinus alpinus (L.). Consequently, the aim of thisstudy was to evaluate the adherent bacterial flora of free-living Arctic charr. Fu rtherm ore,information is neede d as to t he effect, if any, of diet and salinity on the intestinal m icroflora ofthe charr. This is relevant in the context that during recent years land-locked Arctic charrfrom Lake Takvatn have been used as a source for juvenile fish in commercial aquaculture ,even in sea water.This work presents some data on the adherent microflora in the small and large intestinesof free-living Arctic charr from Lake Takvatn, Northern Norway. The study also presentsdata on the intes tinal microflora of wildflsh hat have been transferred to hatchery conditionsand fed either a capelin roe diet or a commercial feed in fresh and sea water for 70 days.

Correspondence: Dr E inar Ring0, University of Trom s0, Institute of Biology and Geology, Dramsveien 201,N-9037 Troms 0, Norway.623


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624 E. Ring0 & E. Str0m

Materials and methodsFishFive immature Arctic charr (40-70 g) were taken in mid August from the littoral zone in LakeTakvatn, Northern Norway. The fish were killed, and immediately placed in ice andtranspo rted to the laboratory . Furth erm ore, free-living charr were transferred to a hatcheryand fed either a 25 mm capelin roe diet or comm ercial feed (Tess Elite 2-5P, Skretting L td,Norway) in aerated fresh and sea water at 8C. The ex periment w as conducted on triplicategroups (25fishper feeding grou p), mean w eight approximately lOOg. A detailed descriptionof the experimental conditions and diets is given elsewhere (Ring0,1991).

Isolation of adherent microorganisms of free-living fishAd here nt bacteria in the gastrointestinal tract were isolated as described elsewhere (Trust &Sparrow 1974; Trust 1975). Briefly, the ventral belly surfaces of five individual fish wereopened to expose the peritoneal cavity. The spleen, gall bladder and liver were removed.Furthermore, fat deposits surrounding the gut were removed. The gastrointestinal tract wasclosed off with sterile clamps as close as possible to the stomach and the vent, then cut freeand transferred to sterile 0-9% saline. Thereafter, the gastrointestinal tract was separatedinto two regions with sterile clamps. Region A com prised the small intestine and region B thelarge intestine. The separate regions were emptied and thoroughly rinsed three times insterile 0-9% saline to remove nonadherent bacteria before homogenation. Homogenationwas carried out in sterile plastic bags on a Stomacher (Seward Laboratory, UK).Hom ogenates of the different regions were diluted in sterile 0-9% saline and 0-1ml volumesof appropriate dilutions were spread on the surface of TSAg plates, which contain TSA(tryptic soy agar) 40g/l and 5g/l glucose. The plates were incubated at 12C and inspecteddaily for up to 4 weeks. After enumeration, a representative selection of colonies weresubcultured on TSAg plates. After confirmation of culture purity the bacteria were thencultured in TSBg medium, which contains TSB (tryptic soy broth) 40g/1 and glucose 5-Og/l.Thereafter, the microorganism cultures were inoculated in glycerol (0-2 ml) and stored at-80C for further identification.

Isolation of microorganisms from faecesIsolation of faecal bacteria was carried out on five randomly chosen fish from each of thefollowing groups: free-living fish caught in Lake Takvatn, and free-living fish held in thehatchery and fed either a capelin roe diet or a commercial feed in either fresh or sea waterafter 70 days of feeding. Stripping consisted of pressing the belly of thefish n the region 2 cmbehind the ventral fins to the anu s. The faecal content from each fish was diluted in sterile0-9% saline, and appropriate dilutions were spread over the surface of TSAg plates.Approx imately 25 colonies from each fish were isolated random ly and tre ated as describedabove.


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Microflora of Arctic charr 625

Identification of bacteriaThe bacteria were classified to the lowest possible taxon by standard biochemical tests(Buchanan & G ibbons 1974; Muroga, Higashi & Keitoku 1987; Stevenson 1989).

Results and discussionTh e gastrointestinal microflora of free-living fishSeveral investigations of intestinal microflora have used methods which isolate only thebacterial flora of the intestinal contents (Newman, Cosenza & Buck 1972; Gilmour,McCallum & Allen 1976; Sakata, O kabayashi & Kakimoto 1980; Ring0 1993 a,b; Sugita etal.1983, 1985, 1987, 1988). However, other studies separate the gastrointestinal tract intodifferent region s, and evaluate t he m icroflora associated with the intestinal wall of thedifferent regions (Trust & Sparrow 1974; Austin & Al-Zahrani 1988). Austin & A l-Zahrani(1988) noted that there was a progressive decline in numbers of aerobic heterotrophicbacteria along the digestive tract of hatchery-reared rainbow trout. However, Trust &Sparrow (1974) showed that numbers of bacteria increased between the oesophagus/stomach/pyloric caeca and the rectum region of free-living salmonids. In the present study,on the other hand , the nu mbe r of microorganisms associated with the wall of the small andlarge intestines of free-living Arctic charr was found to be constant (Table 1).

Table 1. Total viable counts (TVC), total isolates (A/) and bacterial composition (% of total isolates) from thealimentary tract of five individual free-living Arctic charr, Salvelinus alpinus (L.) , from Lake Takvatn, NorthernNorwayRegion*

Bacterial species A BTV C 1-5 X 105 9.5 X iN 109 106Gram-negativeAcinetobacter ip . n.d.f 1-0Aeromonas sp . 19-7 13-3Cytophaga 7-5 2-9Enterobacteriaceae 11-2 13-3Flavobacterium s^ . 9.3 11-4Moraxella sp . 2-8 1-9Pseudomonas sp . 3-7 2-9Vibrio sp . 4-7 4-8Gram-positiveLactobacillus sp. 19-6 23-8

Micrococcus sp . 15'0 11-4Coryneforms 6-5 9-5Streptococcus sp. n.d. 3-8Region A , small intestine; B , large intestine,tn .d . , no t detected.


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The predominant microfiora in the gastrointestinal tract of free-living salmonids fromCanad a consisted of Gram-negative rods with species of Enterobacter sp. , Aeromonas sp . andAcinetobacter sp. present in the greatest numbers (Trust & Sparrow 1974). In the currentstudy, however, Aeromonas sp. , Lactobacillus sp., Micrococcus sp . and En terobacteriaceaewere the predominant bacterial species in both the small and large intestines, whereasAcinetobacter sp. were only present in low numbers in the large intestine (Table 1).

It is generally considered th at Lactobacillus species is present at a high population level inthe gastrointestinal tract of warm-blooded animals (Finegold, Sutter & Mathisen 1983;Conwa y 1989). Howe ver, only a few investigation s have isolated lactic acid bacte ria from thegastrointestinal tract of fish (Schr0der, Clausen, Sandberg & Raa 1980; Str0m 1988; Ring01993a,b). Schroder et al. (1980) isolated a psychrotrophic Lactobacillus plantarum from thegastrointestinal tract of saithe, Pollachius virens (L.). Later, Str0m (1988) demonstrated thatLactobacillus plantarum was the dominant bacterial species in the gastrointestinal tract ofAtlantic salmon, Salmo salar (L.), in the juvenile stages. Recently, studies undertaken onArctic charr have revealed that the population levels of intestinal lactic acid bacteria areaffected by dietary components such as linoleic acid (18:2 n-6) (Ring0 1993a) and chromicoxide (Ring01 993b ). In addition, the present study clearly demon strated th at the populationlevel of Lactobacillus sp. in Arctic charr was affected by both salinity and d iet (Table 2) .

Table 2. Total viable counts (TVC), total isolates (AO from five individual fish and bacterial composition (% of totalisolates) from faeces of free-living fish, and free-living fish fed a capelin roe diet and commercial feed in fresh water(FW) and sea water (SW)

Bacterial speciesTV CNGram-negative

Aeromonas sp .Agrobacterium sp .Alcaligenes sp .EnterobacteriaceaeFlavobacterium sp .Photobacterium sp .Pseudomonas sp .Vibrio sp .

Gram-positiveLactobacillus sp .Micrococctis sp .CoryneformsLeuconostoc sp .Streptococcus sp .

Free-livingfish

1-2 X 10"10527-7n . d . 'n.d.1435-7n.d.2-94-8

21-913-39 5n.d.n.d.

Free-living fish held in hatchery and fedCapelin

FW3-7 X 10*115

17-4n.d.n.d.56-5n.d.4-3n.d.8-78-7n.d.n.d.4-3n.d.

roe dietSW

2-6 X IO'1208-4n.d.n.d.54-2n.d.4-2n.d.

25-0

4-4n.d.n.d.4-2n.d.

1Tess Elite Pluss 2-5 PFW

4-2 X 10'10430-88 6n.d.n,d.4-8n.d.2 9 8

10-6

9 6n.d.n.d.n.d.5-8

SW9-5 X IO'123

4-91227-3n.d.2-4n.d.195

30-8

4-94-9n.d.n.d.3-3

*n.d., not detected.


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Microflora of Arctic charr 627

Table 3 . Total viable counts (TV C), total isolates (N ) an d bacterial composition (% of total isolates) from cape lin ro ediet an d comm ercial feed (Tess Elite Pluss 2 5P)Bacterial species Capelin ro e diet Tess Elite Pluss 2-5 PTV C 3-7 X 10^ 6-7 x 10^N 19 26Agrobacterium sp . 10-5 3-8Alcaligenes sp . 5-2 7-7Enterobacter iaceae n.d. ' 26-9Flavobacterium sp . 10-5 7-7Pseudomonas sp . 31-6 23-0Bacillus sp. 10-5 3-8Micrococcus sp . 5-2 7-7Coryneforms 10-5 . 7-7Streptococcus sp . n.d. 3-8Yeast 15-8 7-7*n,d., no t detected.

Effect of diet and salinity on intestinal microfloraThis study dem onstrated that total viable counts (TVC ) of aerobic microorganisms in faecesfrom free-living fish were substan tially higher (1-2 x 10^) com pare d with wild fish trans ferredto hatchery and fed either a capelin roe diet or commercial feed (abo ut 10'). Moreov er, theTV C value in faeces of charr held in fresh water was higher, by a factor of 10, than that offishheld in sea water (Table 2).It is well known that the intestinal microflora of warm-blooded animals is infiuenced bythe diet (for review see Finegold et al. 1983; Tannock 1983), but information on this topic isscarce in fish (Newman etal. 1972; Sera & Ishida 1972; Sera, Ishida & Katoda 1972). Thisstudy dem on strate d that the inte stinal microflora of the free-living charr differed from that offree-living fish held in a hatchery (Table 2). Aeromonas sp. and Lactobacillus sp .predom inated in the faeces of free-living fish. When the charr were fed on a capelin roe diet,the num ber of these bacterial species decrease d, while the prop ortion of E nterob acteriace aeincreased. The large number of intestinal Enterobacteriaceae isolated from the capeiin roegroup does not originate from the diet, because Enterobacteriaceae were not isolated fromthe diet (Table 3 ). Moreo ver, E nterob acteriac eae were not isolated from free-living fish fedcomm ercial feed. In this rearing group Aeromonas sp . , Pseudomonas sp . , Agrobacterium sp .and Streptococcus sp. formed a high percenta ge of the intestinal microflora.

In addition, the gram-positive bacterial species Bacillus sp. and Coryneforms were notdetected in the faeces of the two rearing groups (Table 2), but the bacterial species wereisolated from both feeds (Table 3). Furthermore, Agrobacterium sp . , Alcaligenes sp . ,Flavobacterium sp. and Micrococcus sp. isolated in the capelin roe diet (Table 3) were notrecovered in faecalia of the capelin roe group (Table 2). We therefore suggest thatAgrobacterium sp . , Alcaligenes sp . , Flavobacterium sp . . Bacillus sp., Coryneforms andMicrococcus sp . are either p resent in low num bers or no t present at all in the digestive tract offree-living A rctic charr transferred to hatchery con ditions.


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Th e differences in intestinal microflora in charr fed the two diets may be due to changes ineither the gut epithelium or attachm ent sites for the gastrointestinal m icroflora, resulting indiffering capability of colonizing the epithelial surface of the small or large intestines.It iswell known that the intestinal microflora of fresh- and seawaterflshharbour differentmicroorganisms. Gene rally, the intestinal microflora of freshwater fish is composed mainly ofAeromonas sp. , Acinetobacter sp., representatives of the family Enterobacteriaceae,Flavobacterium sp., and Pseudomonas sp. (for review see Sakata 1990). However,Lactobacillus sp. constituted a large percentage of the intestinal flora of freshwater-rearedArctic charr (Ring0 1993a,b; this study). This contrasts with the results found for marinefishes, where Vibrio sp . is the dom inant species but Pseudomonas sp. is also isolated in largenumbers (Sakata 1990). This proved to be the case for seawater-reared fish fed on acommercial diet (Table 2). In contrast, the proportion of Enterobacteriaceae, the predomi-nant bacterial species in the capelin roe gro up, was not affected by salinity.

Durin g recent years Takvatn charr have been used as a source of juvenilefishor brood fishin commercial aquaculture. However, use of land-locked charr in commercial aquaculturemay lead to transmission of pathogenic bacteria into hatcheries. According to Histein &Lindstad (1991), infection by atypical Aeromonas salmonicida was observed when Arcticcharr from L ake Takva tn were transferred into a hatchery. This study clearly demonstratedthat Aeromonas sp. was one of the predo minant bacterial species present in the gastrointes-tinal tract and intestinal contents of free-living Arctic charr (Tables 1 and 2). However, thecurrent study gives no information as to whether these bacterial species are Aeromonassalmonicida. It is therefore important in future investigations of the intestinal microflora ofTakv atn charr to evaluate if the Aeromonas sp . present in the gastrointestinal tract is indeedAeromonas salmonicida.

AcknowledgmentThe authors would like to thank Ms Rise Taylor for correction of the English.

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