Einar Ringø

Lactic acid bacteria in fish: a review

Fish are continuously exposed to a wide range of microorganisms present in the environment, and the microbiota of fish have been the subject of several reviews. This review evaluates lactic acid bacteria in fish, and focuses on the several investigations that have demonstrated that Streptococcus, Leuconostoc, Lactobacillus, and Carnobacterium belong to the normal microbiota of the gastrointestinal tract in healthy fish. However, it is well known that the population level of lactic acid bacteria associated with the digestive tract is affected by nutritional and environmental factors like dietary polyunsaturated fatty acids, chromic oxide, stress and salinity. Pathogenic lactic acid bacteria such as Streptococcus, Enterococcus, Lactobacillus, Carnobacterium and Lactococcus have been detected from ascites, kidney, liver, heart and spleen. Some antibiotic treatments and vaccinations have been proposed to cure or prevent these diseases that seem, however, to spread with the development of fish culture. It has also been reported that some lactic acid bacteria isolated from the gastrointestinal tract of fish can act as probiotics. These candidates are able to colonise the gut, and act antagonistic against Gram-negative fish pathogens. These harmless bacteriocin-producing strains may reduce the need to use antibiotics in future aquaculture.

Effects of dietary soyabean meal, inulin and oxytetracycline on intestinal microbiota and epithelial cell stress, apoptosis and proliferation in the teleost Atlantic salmon (Salmo salar L.).

Soyabean meal (SBM)-induced enteritis in the distal intestine of the teleost Atlantic salmon (Salmo salar L.) and other salmonids may be considered a model for diet-related mucosal disorders in other animals and man. The role of the intestinal microbiota in its pathogenesis was explored. Compared to diets containing fishmeal (FM) as the sole protein source, responses to extracted SBM or the prebiotic inulin, with or without oxytetracycline (OTC) inclusion, were studied following a 3-week feeding trial. Intestinal microbiota, organosomatic indices and histology, as well as immunohistochemical detection of proliferating cell nuclear antigen (PCNA), heat shock protein 70 (HSP70) and caspase-3-positive cells in the distal intestine, were studied. Distal intestine somatic indices (DISI) were higher in inulin and lower in SBM compared to FM-fed fish. The low DISI caused by SBM corresponded with histological changes, neither of which was affected by OTC, despite a significant decrease in adherent bacteria count. Image analysis of PCNA-stained sections showed a significant increase in the proliferative compartment length in SBM-fed fish, accompanied by apparent increases in reactivity to HSP70 and caspase-3 along the mucosal folds, indicating induction of cellular repair and apoptosis, respectively. Fish fed the SBM diet had higher total number as well as a more diverse population composition of adherent bacteria in the distal intestine. Thus SBM-induced enteritis is accompanied by induction of distal intestinal epithelial cell protective responses and changes in microbiota. Putative involvement of bacteria in the inflammatory response merits further investigation.

Lipid digestibility and ultrastructural changes in the enterocytes of Arctic char (Salvelinus alpinus L.) fed linseed oil and soybean lecithin

Arctic char (Salvelinus alpinus L.) were fed two isocaloric diets supplemented with 15% linseed oil or soybean lecithin, and subsequently analysed for ultrastructural changes in various intestinal segments (pyloric caeca region, midgut and hindgut) as well as apparent digestibility coefficients (ADC) of the diets. The ADC of dry matter, lipid and individual fatty acids were always highest in fish fed the soybean lecithin diet. In hindgut for example, ADC of lipid and dry matter were 94 and 96%, respectively, in fish fed the soybean lecithin diet compared to 85 and 94% in fish fed linseed oil.There were major ultrastructural differences in the enterocytes between fish fed the two experimental diets. In char fed the linseed oil diet there were substantial accumulations of lipid droplets in the enterocytes from the pyloric caeca and midgut but not in hindgut. These accumulations were not observed in fish fed the soybean lecithin diet. In some cases, deposition of fat in the enterocytes was so great that it resulted in epithelial damage with lipid droplets and cell debris appearing in the intestinal lumen. These changes are likely to be pathological and may lead to intestinal malfunction and represent a major infection route to pathogenic bacteria. On the basis of these results it is suggested that endogenous phospholipid synthesis is insufficient to maintain lipoprotein synthesis in Arctic char when fed diets containing high levels of polyunsaturated fatty acid of total lipid, and that phospholipids should be supplied with such diets.

Prebiotics as immunostimulants in aquaculture: A review

Prebiotics are indigestible fibers that increase beneficial gut commensal bacteria resulting in improvements of the hosts health. The beneficial effects of prebiotics are due to the byproducts generated from their fermentation by gut commensal bacteria. In this review, the direct effects of prebiotics on the innate immune system of fish are discussed. Prebiotics, such as fructooligosaccharide, mannanoligosaccharide, inulin, or β-glucan, are called immunosaccharides. They directly enhance innate immune responses including: phagocytic activation, neutrophil activation, activation of the alternative complement system, increased lysozyme activity, and more. Immunosaccharides directly activate the innate immune system by interacting with pattern recognition receptors (PRR) expressed on innate immune cells. They can also associate with microbe associated molecular patterns (MAMPs) to activate innate immune cells. However, the underlying mechanisms involved in innate immune cell activation need to be further explored. Many studies have indicated that immunosaccharides are beneficial to both finfish and shellfish.

Use of immunostimulants and nucleotides in aquaculture: a review

Currently there are numerous gaps in existing knowledge about exogenous nucleotide application to fish including various aspects of digestion, absorption, metabolism, and influences on various physiological responses, especially expression of immunogenes and modulation of immunoglobulin production. Additional information is also needed in regard to age/size-related responses and appropriate doses and timing of administration. Thus further research in these areas should be pursued. Immunostimulants Abstract It is well established that proper nutrition is essential for maintenance of normal growth and health of all animals including various aquatic species. During the last two decades increased attention has focus on immunostimulants and nucleotides to reduce susceptibility to various stressors and diseases, as well as enhance the overall health of fish. The immune response can be modulated by β-glucans and high-M-alginate. β-glucans are glucose polymers that are major structural components of the cell wall of yeast, fungi, and bacteria, but also of cereals like oat and barley. There is much structural variation in the β-glucans from these different sources, which may influence their physiological functions. Alginate is a polysaccharide composed of β-1,4-D-mannuronic acid (M) and α-L-glucuronic acid (G). In vitro as well as in vivo studies in fish show that especially β-glucans derived from fungi and yeast and alginate have immune modulating properties. Most frequently evaluated are effects on macrophage activation and on lysozyme, respiratory burst and leukocyte activity, which have been suggested to contribute to the increased resistance against infections, after immunostimulant exposure. Although more fish studies are needed, it is tempting to suggest that dietary β-glucans and alginate may be useful tools to prime the host immune system and increase resistance against invading pathogens. As no knowledge is available regarding short versus long-term effects and efficiency, more knowledge is needed on this topic.

The effect of dietary fatty acids on lactic acid bacteria associated with the epithelial mucosa and from faecalia of Arctic charr, Salvelinus alpinus (L.)

Arctic charr, Salvelinus alpinus (L.), held in fresh water, were fed four experimental diets containing different polyunsaturated fatty acids (PUFA). In addition, one group fed a diet containing only coconut oil as sole lipid source served as control. The population of aerobic heterotrophic bacteria associated with the epithelial mucosa and the faecalia was estimated using the dilution plate technique. Generally, the population level of adherent bacteria increased along the digestive tract (stomach, small intestine and large intestine). Adherent Gram‐negative and Gram‐positive bacteria seemed to be present at equal levels in all parts of the alimentary tract. Lactic acid bacteria dominated among the Gram‐positive bacteria, and they were detected in all regions of fish fed the PUFA supplemented diets. The frequency of lactic acid bacteria was highest in the digestive tract of fish fed diets with added 7·0% linolenic acid (18:3 n‐3) or 4% of a PUFA mix. A lower frequency of lactic acid bacteria was found in fish fed dietary linoleic acid (18:2 n‐6), and they were absent or present in low numbers in fish fed the coconut oil diet. It is suggested that dietary fatty acids affect the attachment sites for the gastrointestinal microbiota, possibly by modifying the fatty acid composition of the intestine wall. Numerical taxonomy procedures showed that the lactic acid bacteria Carnobacterium spp. and a Carnobacterium piscicola‐like strain were predominant, with smaller numbers of Lactobacillus plantarum, Streptococcus spp. and Leuconostoc mesenteroides present. Seven strains of Carnobacterium spp. were further identified on the basis of 16S rDNA sequence analysis, and all these strains were identified as Carnobacterium piscicola.

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae.

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot (Scophthalmus maximus) larvae exposed to V. pelagius and/or Aer. caviae. The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 103 ml−1 and 103 larva−1. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 102 bacteria larva−1 on day 3 post‐hatching to approximately 105 bacteria fish−1 16 days post‐hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 104 cfu larva−1 exposed to V. pelagius on days 2, 5 and 8 post‐hatching. However, only 103 of the Vibrio spp. belonged to V. pelagius. When larvae were exposed to Aer. caviae on day 2 post‐hatching, the gut microbiota of 5‐day old larvae was mainly colonized by Aeromonas spp. (104 larva−1), of which 9 × 103 belonged to Aer. caviae. Later in the experiment, at the time when high mortality occurred, 9 × 105Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria (V. pelagius) to the rearing water.

Histological changes in intestine of Atlantic salmon (Salmo salar L.) following in vitro exposure to pathogenic and probiotic bacterial strains

Furunculosis and vibriosis are diseases that cause severe economic losses in the fish-farming industry. The foregut of the Atlantic salmon (Salmo salar L.) was exposed in vitro to two fish pathogens, Aeromonas salmonicida (causative agent of furunculosis) and Vibrio anguillarum (causative agent of vibriosis), and to one probiotic strain, Carnobacterium divergens, at 6 × 104 or 6 × 106 viable bacteria per milliliter. Histological changes following bacterial exposure were assessed by light and electron microscopy. Control samples (foregut exposed to Ringer’s solution only) and samples exposed only to C. divergens had a similar appearance to intact intestinal mucosal epithelium, with no signs of damage. However, exposure of the foregut to the pathogenic bacteria resulted in damaged epithelial cells, cell debris in the lumen, and disorganization of the microvilli. Co-incubation of the foregut with a pathogen and C. divergens did not reverse the damaging effects caused by the pathogen, although these were alleviated when probiotic bacteria were used. Based on these results, we suggest that the probiotic bacterium, C. divergens, is able to prevent, to some extent, pathogen-induced damage in the Atlantic salmon foregut.

Lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.)

The present study reports the effect of excessive handling stress and starvation on the lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.). A relatively low population level (approximately 2 × 103 bacteria per gram wet tissue) of viable adherent heterotrophic bacteria was associated with the digestive tract (foregut, midgut and hindgut). Of the 752 bacterial isolates isolated from diet, water and the digestive tract, 201 isolates belonged to the carnobacteria. Of these isolates, one from the diet, one from the rearing water and 80 from the gastrointestinal tract, were further identified on the basis of 16S rDNA sequence analysis. All these isolates were identified as being Carnobacterium piscicola‐like. Daily repeated stress and starvation of the fish over 11 d had no influence on the total culturable bacterial numbers or population level of C. piscicola associated with the digestive tract. C. piscicola‐like isolates colonizing the various intestinal regions (foregut, midgut and hindgut) were also screened for their ability to produce growth inhibitory compounds active against the fish pathogen Aeromonas salmonicida. Of the 199 C. piscicola isolates tested, 139 inhibited growth of the pathogen.

Damaging effect of the fish pathogen Aeromonas salmonicida ssp. salmonicida on intestinal enterocytes of Atlantic salmon (Salmo salar L.)

In fish, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) gastrointestinal tract. Bacteria can cross the gastrointestinal lining in three different ways. In undamaged tissue, bacteria can translocate by transcellular or paracellular routes. Alternatively, bacteria can damage the intestinal lining with extracellular enzymes or toxins before entering. Using an in vitro (Ussing chamber) model, this paper describes intestinal cell damage in Atlantic salmon (Salmo salar L.) caused by the fish pathogen Aeromonas salmonicida ssp. salmonicida, the causative agent of furunculosis. The in vitro method clearly demonstrated substantial detachment of enterocytes from anterior region of the intestine (foregut) upon exposure to the pathogen. In the hindgut (posterior part of the intestine), little detachment was observed but cellular damage involved microvilli, desmosomes and tight junctions. Based on these findings, we suggest that A. salmonicida may obtain entry to the fish by seriously damaging the intestinal lining. Translocation of bacteria through the foregut (rather than the hindgut) is a more likely infection route for A. salmonicida infections in Atlantic salmon.