Aquaculture and Fisheries Management 1994, 25, 623-629

Microfiora of Arctic charr, Salvelinus alpinus (L.): gastrointestinal microflora of free-living fish and effect of diet and salinity on intestinal microflora
E. RING0 University of Troms0, Institute of Biology and Geology, Troms0, Norway E. STR0M Troms0 College of Health Care Education, Troms0, Norway

Abstract. The adherent aerobic bacterial flora present in the gastrointestinal tract and faeces of free-living Arctic charr, Salvelinus alpinus (L.), from Lake Takvatn, Northern Norway, were identified both qualitatively and quantitatively. Approximately 10^ bacteria g~' were found in both the small and large intestines. The predominant bacterial species were identified as Aeromonas, Enterobacteriaceae, Micrococcus and Lactobacillus. Other microorganisms isolated included Acinetobacter, Cytophaga, Flavobacterium, Moraxella, Pseudomonas, Vibrio, Coryneforms and Streptococcus. The intestinal microflora of free-living fish was dominated by Aeromonas and Lactobacillus, but the intestinal bacterial flora of wild fish transferred to hatchery was affected by feeding them either a capelin roe diet or a commercial feed in fresh and sea water. Approximately 55% ofthe bacterial flora in intestinal contents from fish fed the capelin roe diet was Enterobacteriaceae when the fish were held in fresh and sea water. However, when the wild-caught charr were fed a commercial diet in fresh water, Aeromonas and Pseudomonas dominated in faeces, while Vibrio and Pseudomoruis were predominant in the diet group held in sea water.

Introduction

Studies have been conducted on the bacterial flora associated with the intestinal wall in different 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 the gastrointestinal tract of Arctic charr, Salvelinus alpinus (L.). Consequently, the aim of this study was to evaluate the adherent bacterial flora of free-living Arctic charr. Furthermore, information is needed as to the effect, if any, of diet and salinity on the intestinal microflora of the charr. This is relevant in the context that during recent years land-locked Arctic charr from 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 intestines of free-living Arctic charr from Lake Takvatn, Northern Norway. The study also presents data on the intestinal microflora of wildflshthat have been transferred to hatchery conditions and fed either a capelin roe diet or a commercial feed in fresh and sea water for 70 days.

Correspondence: Dr Einar Ring0, University of Troms0, Institute of Biology and Geology, Dramsveien 201, N-9037 Troms0, Norway. 623

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E. Ring0 & E. Str0m

Materials and methods Fish Five immature Arctic charr (40-70 g) were taken in mid August from the littoral zone in Lake Takvatn, Northern Norway. The fish were killed, and immediately placed in ice and transported to the laboratory. Furthermore, free-living charr were transferred to a hatchery and fed either a 25 mm capelin roe diet or commercial feed (Tess Elite 2-5P, Skretting Ltd, Norway) in aerated fresh and sea water at 8C. The experiment was conducted on triplicate groups (25fishper feeding group), mean weight approximately lOOg. A detailed description of the experimental conditions and diets is given elsewhere (Ring0,1991).

Isolation of adherent microorganisms of free-living fish Adherent bacteria in the gastrointestinal tract were isolated as described elsewhere (Trust & Sparrow 1974; Trust 1975). Briefly, the ventral belly surfaces of five individual fish were opened to expose the peritoneal cavity. The spleen, gall bladder and liver were removed. Furthermore, fat deposits surrounding the gut were removed. The gastrointestinal tract was closed off with sterile clamps as close as possible to the stomach and the vent, then cut free and transferred to sterile 0-9% saline. Thereafter, the gastrointestinal tract was separated into two regions with sterile clamps. Region A comprised the small intestine and region B the large intestine. The separate regions were emptied and thoroughly rinsed three times in sterile 0-9% saline to remove nonadherent bacteria before homogenation. Homogenation was carried out in sterile plastic bags on a Stomacher (Seward Laboratory, UK). Homogenates of the different regions were diluted in sterile 0-9% saline and 0-1 ml volumes of 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 inspected daily for up to 4 weeks. After enumeration, a representative selection of colonies were subcultured on TSAg plates. After confirmation of culture purity the bacteria were then cultured 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 faeces Isolation of faecal bacteria was carried out on five randomly chosen fish from each of the following groups: free-living fish caught in Lake Takvatn, and free-living fish held in the hatchery and fed either a capelin roe diet or a commercial feed in either fresh or sea water after 70 days of feeding. Stripping consisted of pressing the belly of thefishin the region 2 cm behind the ventral fins to the anus. The faecal content from each fish was diluted in sterile 0-9% saline, and appropriate dilutions were spread over the surface of TSAg plates. Approximately 25 colonies from each fish were isolated randomly and treated as described above.

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625

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

Results and discussion The gastrointestinal microflora of free-living fish Several investigations of intestinal microflora have used methods which isolate only the bacterial flora of the intestinal contents (Newman, Cosenza & Buck 1972; Gilmour, McCallum & Allen 1976; Sakata, Okabayashi & Kakimoto 1980; Ring01993a,b; Sugita etal. 1983, 1985, 1987, 1988). However, other studies separate the gastrointestinal tract into different regions, and evaluate the microflora associated with the intestinal wall of the different regions (Trust & Sparrow 1974; Austin & Al-Zahrani 1988). Austin & Al-Zahrani (1988) noted that there was a progressive decline in numbers of aerobic heterotrophic bacteria 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 number of microorganisms associated with the wall of the small and large 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 the alimentary tract of five individual free-living Arctic charr, Salvelinus alpinus (L.), from Lake Takvatn, Northern Norway Region* Bacterial species TVC N Gram-negative Acinetobacter ip. Aeromonas sp. Cytophaga Enterobacteriaceae Flavobacterium s^. Moraxella sp. Pseudomonas sp. Vibrio sp. Gram-positive Lactobacillus sp. Micrococcus sp. Coryneforms Streptococcus sp. Region A, small intestine; B, large intestine, tn.d., not detected. A 1-5 X 105 109 n.d.f 19-7 7-5 11-2 9.3 2-8 3-7 4-7 19-6 15'0 6-5 n.d. B 9.5 X i 106 1-0 13-3 2-9 13-3 11-4 1-9 2-9 4-8 23-8 11-4 9-5 3-8

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The predominant microfiora in the gastrointestinal tract of free-living salmonids from Canada consisted of Gram-negative rods with species of Enterobacter sp., Aeromonas sp. and Acinetobacter sp. present in the greatest numbers (Trust & Sparrow 1974). In the current study, however, Aeromonas sp., Lactobacillus sp., Micrococcus sp. and Enterobacteriaceae were the predominant bacterial species in both the small and large intestines, whereas Acinetobacter sp. were only present in low numbers in the large intestine (Table 1). It is generally considered that Lactobacillus species is present at a high population level in the gastrointestinal tract of warm-blooded animals (Finegold, Sutter & Mathisen 1983; Conway 1989). However, only a few investigations have isolated lactic acid bacteria from the gastrointestinal tract of fish (Schr0der, Clausen, Sandberg & Raa 1980; Str0m 1988; Ring0 1993a,b). Schroder et al. (1980) isolated a psychrotrophic Lactobacillus plantarum from the gastrointestinal tract of saithe, Pollachius virens (L.). Later, Str0m (1988) demonstrated that Lactobacillus plantarum was the dominant bacterial species in the gastrointestinal tract of Atlantic salmon, Salmo salar (L.), in the juvenile stages. Recently, studies undertaken on Arctic charr have revealed that the population levels of intestinal lactic acid bacteria are affected by dietary components such as linoleic acid (18:2 n-6) (Ring0 1993a) and chromic oxide (Ring01993b). In addition, the present study clearly demonstrated that the population level of Lactobacillus sp. in Arctic charr was affected by both salinity and diet (Table 2).

Table 2. Total viable counts (TVC), total isolates (AO from five individual fish and bacterial composition (% of total isolates) 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) Free-living fish held in hatchery and fed1 FreeBacterial species living fish 1-2 X 10" Capelin roe diet Tess Elite Pluss 2-5 P

FW
3-7 X 10*

SW
2-6 X IO'

FW
4-2 X 10'

SW
9-5 X IO'

TVC N
Gram-negative Aeromonas sp. Agrobacterium sp. Alcaligenes sp. Enterobacteriaceae Flavobacterium sp. Photobacterium sp. Pseudomonas sp. Vibrio sp. Gram-positive Lactobacillus sp. Micrococctis sp. Coryneforms Leuconostoc sp. Streptococcus sp. *n.d., not detected.

105
27-7 n.d.' n.d.

115
17-4 n.d. n.d. 56-5 n.d.

120 8-4
n.d. n.d. 54-2 n.d.

104
30-8

123 4-9 122 7-3

n.d.

86
n.d. n,d.

143 5-7
n.d.

4-8
n.d.

2-4
n.d.

4-3
n.d.

4-2
n.d. 25-0

2-9 4-8
21-9 13-3

298
10-6

195
30-8

8-7 8-7
n.d. n.d.

4-4
n.d. n.d.

96
n.d. n.d. n.d.

4-9 4-9
n.d. n.d.

95
n.d. n.d.

4-3
n.d.

4-2
n.d.

5-8

3-3

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627

Table 3. Total viable counts (TVC), total isolates (N) and bacterial composition (% of total isolates) from capelin roe diet and commercial feed (Tess Elite Pluss 2 5P) Bacterial species TVC N Agrobacterium sp. Alcaligenes sp. Enterobacteriaceae Flavobacterium sp. Pseudomonas sp. Bacillus sp. Micrococcus sp. Coryneforms Streptococcus sp. Yeast *n,d., not detected. Capelin roe diet 3-7 X 10^ 19 10-5 5-2 n.d.' 10-5 31-6 10-5 5-2 10-5 n.d. 15-8 Tess Elite Pluss 2-5 P 6-7 x 10^ 26 3-8 7-7 26-9 7-7 23-0 3-8 7-7 7-7 3-8 7-7

Effect of diet and salinity on intestinal microflora This study demonstrated that total viable counts (TVC) of aerobic microorganisms in faeces from free-living fish were substantially higher (1-2 x 10^) compared with wild fish transferred to hatchery and fed either a capelin roe diet or commercial feed (about 10'). Moreover, the TVC value in faeces of charr held in fresh water was higher, by a factor of 10, than that offish held in sea water (Table 2). It is well known that the intestinal microflora of warm-blooded animals is infiuenced by the diet (for review see Finegold et al. 1983; Tannock 1983), but information on this topic is scarce in fish (Newman etal. 1972; Sera & Ishida 1972; Sera, Ishida & Katoda 1972). This study demonstrated that the intestinal microflora of the free-living charr differed from that of free-living fish held in a hatchery (Table 2). Aeromonas sp. and Lactobacillus sp. predominated in the faeces of free-living fish. When the charr were fed on a capelin roe diet, the number of these bacterial species decreased, while the proportion of Enterobacteriaceae increased. The large number of intestinal Enterobacteriaceae isolated from the capeiin roe group does not originate from the diet, because Enterobacteriaceae were not isolated from the diet (Table 3). Moreover, Enterobacteriaceae were not isolated from free-living fish fed commercial feed. In this rearing group Aeromonas sp., Pseudomonas sp., Agrobacterium sp. and Streptococcus sp. formed a high percentage of the intestinal microflora. In addition, the gram-positive bacterial species Bacillus sp. and Coryneforms were not detected in the faeces of the two rearing groups (Table 2), but the bacterial species were isolated from both feeds (Table 3). Furthermore, Agrobacterium sp., Alcaligenes sp., Flavobacterium sp. and Micrococcus sp. isolated in the capelin roe diet (Table 3) were not recovered in faecalia of the capelin roe group (Table 2). We therefore suggest that Agrobacterium sp., Alcaligenes sp., Flavobacterium sp.. Bacillus sp., Coryneforms and Micrococcus sp. are either present in low numbers or not present at all in the digestive tract of free-living Arctic charr transferred to hatchery conditions.

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E. Ring0 & E. Str0m

The differences in intestinal microflora in charr fed the two diets may be due to changes in either the gut epithelium or attachment sites for the gastrointestinal microflora, resulting in differing capability of colonizing the epithelial surface of the small or large intestines. It is well known that the intestinal microflora of fresh- and seawaterflshharbour different microorganisms. Generally, the intestinal microflora of freshwater fish is composed mainly of Aeromonas 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-reared Arctic charr (Ring0 1993a,b; this study). This contrasts with the results found for marine fishes, where Vibrio sp. is the dominant species but Pseudomonas sp. is also isolated in large numbers (Sakata 1990). This proved to be the case for seawater-reared fish fed on a commercial diet (Table 2). In contrast, the proportion of Enterobacteriaceae, the predominant bacterial species in the capelin roe group, was not affected by salinity. During recent years Takvatn charr have been used as a source of juvenilefishor brood fish in commercial aquaculture. However, use of land-locked charr in commercial aquaculture may lead to transmission of pathogenic bacteria into hatcheries. According to Histein & Lindstad (1991), infection by atypical Aeromonas salmonicida was observed when Arctic charr from Lake Takvatn were transferred into a hatchery. This study clearly demonstrated that Aeromonas sp. was one of the predominant bacterial species present in the gastrointestinal tract and intestinal contents of free-living Arctic charr (Tables 1 and 2). However, the current study gives no information as to whether these bacterial species are Aeromonas salmonicida. It is therefore important in future investigations of the intestinal microflora of Takvatn charr to evaluate if the Aeromonas sp. present in the gastrointestinal tract is indeed Aeromonas salmonicida.

Acknowledgment

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

References
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