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Intensive fish production worldwide has increased the risk of infectious diseases. However, before any infection can be established, pathogens must penetrate the primary barrier. In fish, the three major routes of infection are the skin, gills and gastrointestinal (GI) tract. The GI tract is essentially a muscular tube lined by a mucous membrane of columnar epithelial cells that exhibit a regional variation in structure and function. In the last two decades, our understanding of the endocytosis and translocation of bacteria across this mucosa, and the sorts of cell damage caused by pathogenic bacteria, has increased. Electron microscopy has made a valuable contribution to this knowledge. In the fish-farming industry, severe economic losses are caused by furunculosis (agent, Aeromonas salmonicida spp. salmonicida) and vibriosis [agent, Vibrio ( Listonella) anguillarum]. This article provides an overview of the GI tract of fish from an electron microscopical perspective focusing on cellular damage (specific attack on tight junctions and desmosomes) caused by pathogenic bacteria, and interactions between the ‘good’ intestinal bacteria [e.g. lactic acid bacteria (LAB)] and pathogens. Using different in vitro methods, several studies have demonstrated that co-incubation of Atlantic salmon ( Salmo salar L.) foregut (proximal intestine) with LAB and pathogens can have beneficial effects, the cell damage caused by the pathogens being prevented, to some extent, by the LAB. However, there is uncertainty over whether or not similar effects are observed in other species such as Atlantic cod ( Gadus morhua L.). When discussing cellular damage in the GI tract of fish caused by pathogenic bacteria, several important questions arise including: (1) Do different pathogenic bacteria use different mechanisms to infect the gut? (2) Does the gradual development of the GI tract from larva to adult affect infection? (3) Are there different infection patterns between different fish species? The present article addresses these and other questions. [ABSTRACT FROM AUTHOR]

Abstract: The digestive tract of fish is essentially a muscular tube lined by a mucous membrane of columnar epithelial cells that exhibit regional variation in structure and function. In the last two decades, our understanding of translocation of bacteria in the fish gut has increased, and electron microscopy has contributed to this knowledge. Translocation is the movement of viable and non-viable bacteria and bacterial products (such as endotoxin) from the intestinal lumen through the epithelial mucosa to infect otherwise sterile tissues. This paper provides an overview of the gastrointestinal tract of larvae and fry from an electron microscopical perspective focussing on factors (mucus, interactions between probiotics and pathogens, and stress) affecting translocation of bacteria (transcellular and paracellular), cellular damage (specific attack on tight junctions and desmosomes caused by pathogenic bacteria) and pathogenesis. Furthermore, the paper will present information on the occurrence and translocation of pathogenic bacteria in the gastrointestinal tract of larvae, fry and adult fish. Currently, it is generally accepted that the major routes of infection in fish are through the skin, gills and gastrointestinal tract. As the gastrointestinal tract is a potential port of entry for pathogens, the use of electron microscopy in studies on microbiota in fish digestive tract is a valuable method for increasing our understanding of the mechanisms involved in entry of the pathogens. Possible mechanisms involved in translocation of bacteria in fish gastrointestinal tract, autochthonous (indigenous) and pathogenic, are discussed. [Copyright &y& Elsevier]

The primary aim of the present study was to evaluate the population level of adherent (autochthonous) aerobic and facultative anaerobic bacteria in the hindgut of healthy Arctic charr ( Salvelinus alpinus L.) fed dextrin or inulin. This was assessed by the dilution plate technique, and visualized using both transmission and scanning electron microscopy. A population level of 4.8 × 105 adherent bacteria per gram wet mass was found in the hindgut of fish fed a casein-based diet supplemented with 15% dextrin. However, substituting dextrin with 15% inulin reduced the bacterial population level in the hindgut (3.56 × 104). A total of 217 bacterial isolates were identified by key phenotypical and biochemical characteristics. In addition, 22 strains were also identified by partial sequencing of the 16S rRNA gene. The composition of bacteria colonizing the hindgut of Arctic charr fed dextrin was dominated by the genera Staphylococcus, Pseudomonas, Micrococcus, Psychrobacter glacincola and Streptococcus. However, bacteria colonizing the hindgut of fish fed inulin were dominated by Gram-positive bacteria of the genera Staphylococcus, Streptococcus, Carnobacterium and Bacillus. While Carnobacterium divergens-like strains were isolated from charr fed dextrin, Carnobacterium maltaromicus-like strains were isolated from the hindgut of fish fed inulin. Electron microscopical analysis of hindgut regions confirmed traditional culture-based microbial analysis as fewer bacterial cells were observed between microvilli and associated with the surfaces of enterocytes of fish fed inulin rather than dextrin. [ABSTRACT FROM AUTHOR]

Abstract: Populations of aerobic heterotrophic bacteria present in the gastrointestinal (GI) tract of healthy Atlantic cod (Gadus morhua L.) fed three different diets, fish meal, standard or a bioprocessed soybean meal (BPSBM), were estimated using the dilution plate technique. A total of 944 isolates were characterised by biochemical and physiological properties and 425 isolates were identified further by sequencing the 16S rRNA genes. Our results showed that gut microbiota were affected by dietary manipulation. The GI tract of fish fed fish meal was dominated by Gram-positive bacteria of the genera Brochothrix and Carnobacterium. The Gram-negative bacteria Chryseobacterium spp. and Psychrobacter glacincola, and Gram-positive bacteria belonging to Carnobacterium, dominated in the digestive tract of fish fed soybean meal. In contrast to these results, genus Psychrobacter dominated in the GI tract when fish were fed BPSBM. Until recently, it was generally suggested that the gut microbiota of fish were less diverse than in homoeothermic animals. However, the present study identified several “new” bacterial species isolated from the alimentary tract of Atlantic cod. These “new” bacterial species are not normally isolated from the GI tract of fish. Based on our finding we suggest that the GI tract microbiota of fish might not be as simple as believed. Antagonistic activity of carnobacteria regarding inhibition of growth of two fish pathogens (Aeromonas salmonicida ssp. salmonicida and Vibrio anguillarum) was observed. However, some difference in the antibacterial activity of Carnobacterium spp. was observed. Whether this antagonistic activity has any effect in challenge studies will be discussed, especially in relation to the finding that the digestive tract is one of the major infection routes in fish. [Copyright &y& Elsevier]

Abstract: Replicate groups of Atlantic cod were fed 5 diets in which fish meal protein was replaced with 25% (25 PP), 50% (50 PP), 75% (75 PP) and 100% (100 PP) of a plant protein mixture containing 50% wheat gluten, 36% soy protein concentrate and 14% bioprocessed soybean meal. After 28 weeks, fish were sacrificed and analysed for growth, feed conversion, nutrient digestibility (see [Hansen, A.-C., Rosenlund, G., Karlsen, Ø., Koppe, W., Hemre, G.-I., Accepted for publication. Total replacement of fish meal with plant proteins in diets for Atlantic cod (Gadus morhua L.) I: Effects on growth and protein retention. Aquaculture]) and health status. Except for small reductions in the mean cell volume and haemoglobin content of red blood corpuscles, health indicators in blood and plasma were mostly unaffected by diet. Although the apparent digestibility coefficient (ADC) of nutrients was only marginally affected by diet [Hansen, A.-C., Rosenlund, G., Karlsen, Ø., Koppe, W., Hemre, G.-I., Accepted for publication. Total replacement of fish meal with plant proteins in diets for Atlantic cod (Gadus morhua L.) I: Effects on growth and protein retention. Aquaculture], faeces moisture content increased non-linearly peaking in fish fed the 100 PP diet where dry matter was only 11% compared to 18% in the control fish. This indicates a progression towards a diarrhoea-like condition in 100 PP fish. Feeding plant proteins did not affect gut relative weight but did affect intestinal morphology. In particular, there was a general increase in cellularity of the lamina propria and a modest goblet cell hypertrophy and hyperplasia which peaked in fish fed the 100 PP diet. More severe enteritis-like conditions were rare and found in only two fish fed the 100 PP diet. The dietary effect on goblet cells may be caused by phytate or fibres rather than the primary enteritis-inducing compounds found in untreated soybean meal. Hindgut segments seemed to be more affected than midgut thus suggesting an up-concentration of the causative agents of intestinal alteration in hindgut regions. Soyasaponins were not degraded during gut transit although the various conjugates were hydrolysed increasing the level of un-conjugated soyasaponins in the faeces. The intestinal level of heat shock protein 70 (HSP70) was unaffected by diet except in one replicate pen of fish fed the 100 PP diet where expression increased fourfold compared to the other replicate and other diets. The reason for this remains obscure. In conclusion, the plant ingredients used in the present study appear not to affect cod health to any major degree when used to replace up to 75% of fish meal protein. An increase to 100% may induce enteritis-like conditions in the guts of some fish and also activate HSP70 stress-gene mRNA expression in the intestine. [Copyright &y& Elsevier]

Abstract: Groups of rainbow trout (Oncorhynchus mykiss Walbaum) in feeding (guts filled with digesta) or food-deprived (3 days of diet deprivation) states were subjected to 15 min of acute stress. Blood samples and intestinal tissue were collected and prepared for physiological, chemical and ultrastructural analysis immediately before stress, and at 4 and 48 h post-stress. Haematocrit, plasma cortisol and lactate levels increased following stress, and the response appeared to be more pronounced in food-deprived than in fed fish. Plasma glucose appeared to increase more in fed fish. Stress did not seem to cause massive tissue damage as measured by cellular leakage of transaminase enzymes into the blood. Furthermore, the plasma oxidative stress marker malondialdehyde did not increase markedly following stress. The content of malondialdehyde did not change following stress neither in midgut nor hindgut sections, and nor did membrane lipid class and fatty acid compositions. Ultrastructural studies showed that stress caused significant widening of the tight junctions between enterocytes in the midgut, with little effect seen in the hindgut. Fed fish appeared to experience more damage than food-deprived fish as judged by the ultrastructural analysis. But these changes were to a large extent transient and cellular organization in the midgut had returned to normal values within 2 days. Intestinal paracellular permeability of the midgut increased 4 h post-stress and was further increased 48 h post-stress of food-deprived fish, whereas no effect was seen in fed trout. In hindgut of food-deprived fish, the permeability appeared to increase sometime after 4 h and was still elevated 48 h after stress. No change in permeability occurred in fed fish. The adherent microbial population level and composition in hindgut was significantly reduced following stress, while the level increased in faeces. This suggests that substantial amounts of mucus are peeled off following stress. It is concluded that, under the present experimental conditions, acute stress causes cellular alteration in rainbow trout gastrointestinal tract. Ultrastructural damages are mainly observed in midgut, but most changes appear to be transient returning to normal levels within 48 h. Changes in adherent intestine microbial population level and compositions together with increased intestinal paracellular permeability following acute stress suggest a possible increased susceptibility to bacterial infections after stress and that active feeding may have a protective role. [Copyright &y& Elsevier]