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Cell Tissue Res (2004) 318: 305–311 DOI 10.1007/s00441-004-0934-2 REGULAR A RTICLE Einar Ringø . Fredrik Jutfelt . Premasany Kanapathippillai . Yvonne Bakken . Kristina Sundell . Johan Glette . Terry M. Mayhew . Reidar Myklebust . Rolf Erik Olsen Damaging effect of the fish pathogen Aeromonas salmonicida ssp. salmonicida on intestinal enterocytes of Atlantic salmon (Salmo salar L.) Received: 13 November 2003 / Accepted: 26 May 2004 / Published online: 3 August 2004 # Springer-Verlag 2004 Abstract 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 Financial support from the Commission of the European Communities, quality of Life and Management of Living Resources programme, project Q5RT-2000-31656 “Gastrointestinal Functions and Food Intake Regulation in Salmonids: Impact of Dietary vegetable Lipids” (GUTINTEGRITY) and from Magnus Bergvalls Stiftelse for KS, is acknowledged. This work does not represent the opinion of the European Community, which is thus not responsible for any use of the data presented. E. Ringø (*) . Y. Bakken Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, 9292 Tromsø, Norway e-mail: [email protected] Fax: +47-77-694911 E. Ringø . J. Glette Institute of Marine Research, Bergen, Norway F. Jutfelt . K. Sundell Department of Zoology, University of Gothenburg, Göteborg, Sweden 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. Keywords Ussing chamber . Fish digestive tract . Aeromonas . Cell damage . Electron microscopy . Salmo salar (Teleostei) Introduction Intensive fish production has increased the risk of infectious diseases all over the world (Press and Lillehaug 1995; Karunasagar and Karunasagar 1999), but to prevent P. Kanapathippillai Department of Electron Microscopy, Faculty of Medicine, University of Tromsø, Tromsø, Norway Y. Bakken Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, Tromsø, Norway T. M. Mayhew School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, UK R. Myklebust Institute of Anatomy and Cell Biology, University of Bergen, Bergen, Norway R. E. Olsen Institute of Marine Research, Matre Aquaculture Research Station, Matredal, Norway 306 microbial entry, fish have various protective mechanisms to hinder translocation of pathogens across the primary barriers. These include production of mucus by goblet cells, the apical acidic microenvironment of the intestinal epithelium, cell turnover, peristalsis, gastric acidity, lysozyme and antibacterial activity of epidermal mucus. At the same time, pathogenic microorganisms have evolved mechanisms to penetrate these barriers. The three major routes of infection are: (a) skin (for review, see Birkbeck and Ringø 2004), (b) gills (for review, see Birkbeck and Ringø 2004), and (c) the gastrointestinal (GI) tract (Sakai 1979; Rose et al. 1989; Chair et al. 1994; Grisez et al. 1996; Olsson et al. 1996; Romalde et al. 1996; Jöborn et al. 1997; Robertson et al. 2000; Lødemel et al. 2001). During the last decade, our knowledge of bacterial adhesion (at molecular and genetic levels) in endothermic animals has increased, and electron microscopy has contributed significantly to this knowledge (Knutton 1995). Although several papers have described pathogenesis in fish (Birkbeck and Ringø 2004), few investigations have used electron microscopy (transmission and/or scanning) to evaluate the effect of bacterial infection on morphology in the GI tract of fish (Ringø et al. 2003). Historically, Aeromonas salmonicida ssp. salmonicida (A. salmonicida) has been recognised as the most important bacterial salmonid pathogen because of its severe economic impact especially on the aquaculture industry (Olivier 1997). Ever since the Furunculosis Committee considered the intestine as a valuable location for isolation of the microorganism (Mackie et al. 1930), controversy has existed as to whether or not the intestine can function as an infection route. However, the presence of A. salmonicida in intestinal samples from several fish species (Ringø et al. 1997; Lødemel et al. 2001; Petersen and Dalsgaard 2003), together with evidence that salmonids fed diets containing probiotic bacteria or soybean meal showed changes in mortality rate after cohabitant challenge with A. salmonicida (Krogdahl et al. 2000; Robertson et al. 2000), indicates that the intestine can be an important route of infection. It is known that pathogenic bacteria produce a wide array of virulent factors (including haemolysins, cytotoxins, enterotoxins, endotoxins and adhesins), which can affect the intestinal barrier function and facilitate translocation (Chopra et al. 2000). Translocation mechanisms include increased receptor-mediated endocytosis (Skirpstunas and Baldwin 2002), increased paracellular permeability mediated by effects on junctional complexes and the cytoskeleton, and direct damage to the intestinal cells (Fasano 2002). The aim of the present study was to investigate, using an in vitro model, whether exposure of the intestinal mucosa of the salmonid fish Atlantic salmon (Salmo salar L.) to A. salmonicida ssp. salmonicida affects the morphology of the intestinal epithelium. Materials and methods Fish Unvaccinated Atlantic salmon (Salmo salar L.) of the Norwegian Breeding Programme were maintained in freshwater under continuous light regimes from hatching at the Institute of Marine Research (IMR), Matre Aquaculture Research Station, Matredal, Norway until an average weight of 73 g was achieved. They were then transferred to the disease challenge laboratory in Bergen where they were maintained in 150 l tanks each holding 100 fish. The water temperature was 10°C and the water flow 10 l/min. Fish were left to adapt to the new conditions for 2 weeks before initiation of the experiments. From initial feeding to the end of the experiment, they were fed to excess using 24-h disc feeders loaded with commercial diets suitable to their relative size (EWOS Innovation Ltd). The experimental protocols and procedures were approved by the Animal Use and Care Committee at IMR, Bergen, Norway. Bacteria Aeromonas salmonicida ssp. salmonicida strain VI88/09/03175 (culture collection, Central Veterinary Laboratory, Oslo, Norway) was used in all experiments. This strain is pathogenic to salmonids (Samuelsen et al. 1998). The bacterium was stored in cryo tubes with glycerol at −80°C, and was inoculated into brain–heart infusion medium (BHI) (Merck, Germany). After 24 h, 1% of this culture was inoculated into a new culture and incubated for another 24 h. This suspension was adjusted to a transmission at 520 nm (T520) of 3.5 by adding BHI. The bacteria were washed four times in PBS (recipe), resuspended in 10 ml salmon Ringer solution (NaCl 140 mM; KCl 2.5 mM; CaCl2 1.5 mM; MgSO4 0.8 mM; glucose 10 mM and HEPES 5 mM, pH was adjusted to 7.8 with 1.5 mM TRIS base) and stored refrigerated until use. Viability of bacteria was tested by plating out bacterial suspensions on brain–heart infusion agar with 1.5% NaCl. More than 90% of the bacteria were viable. Ussing chamber and in vitro exposure Fish raised at IMR were killed by a blow to the head, and the intestine (from just posterior to the attachment of the pyloric caeca to the anus) was carefully removed into salmon Ringer solution, continuously gassed with air and kept on ice. The intestines were separated into two parts. The anterior part or foregut (from just posterior to the pyloric caeca to the ileorectal valve) and the posterior part or hindgut (from ileorectal valve to anus). The intestinal segments were cut open along a mesenteric border and rinsed in ice-cold salmon Ringer solution. Intestinal segments were mounted in Ussing chambers (Grass and Sweetana 1988) modified according to Sundell 307 et al. (2003). Chambers were filled with salmon Ringer solution and the temperature was kept at 10°C by a cooling mantle. Mixing and oxygenation was achieved by gas-lift with air. The exposed tissue surface area was 0.75 cm2 and the half-chamber volume 5 ml. The chambers were equipped with one pair of platinum electrodes for current passage and one pair of Ag/AgCl electrodes (Radiometer, Copenhagen) for measurement of transepithelial potential differences (TEP) and further calculation of transepithelial resistance (TER) and short circuit current (SCC), as described by Sundell et al. (2003). All preparations were allowed 60 min of equilibration before the experiments started. Exposure of the intestinal mucosa to A. salmonicida was initiated by changing the Ringer solution in the mucosal compartment to salmon Ringer containing 6.2×106 bacteria/ml and the serosal compartment to fresh salmon Ringer solution. The three electrical parameters TEP, TER and SCC were monitored continuously throughout the experiment and used to control preparation viability. Fig. 1 Transmission electronmicroscopy (TEM) micrograph of the foregut (anterior part of the intestine) of Atlantic salmon (Salmo salar L.), exposed to dextran containing Ringer (not exposed to bacteria) showing normal enterocytes. L Lumen, MV microvilli, JC junctional complex, M mitochondria, N nucleolus. ×3750. Bar: 5 μm Electron-microscopical sampling Intestinal segments exposed to A. salmonicida, and segments not exposed to the pathogen, were immediately fixed in McDowell’s fixative (McDowell and Trump 1976) and prepared for analyses by transmission electron microscopy (TEM) as described elsewhere (Ringø et al. 2001). Microbial examinations After 8 weeks of feeding, immediately prior to the Ussing chamber experiment, five fish were killed by a sharp blow on the head. The digestive tract was divided into foregut and hindgut, and adherent gut bacteria were isolated from the two regions as described elsewhere (Ringø and Olsen 1999). Homogenates of the foregut and hindgut were diluted in sterile 0.9% saline and appropriate dilutions were spread on the surface of tryptic soy agar (Difco) plates added 5% glucose and 1% NaCl. The plates were incubated at 12°C and inspected regularly for up to 4 weeks. After enumeration (Ringø and Olsen 1999), a 308 representative selection of colonies (49) was subcultured until purity was achieved. Whenever necessary, the isolates were tested for up to 52 biochemical and physiological properties as described by Ringø and Olsen (1999). Results TEM examination of the foregut of Atlantic salmon not exposed to the fish pathogenic bacteria, A. salmonicida, using the in vitro approach, i.e. the Ussing chamber method, showed that the mucosa appeared normal with an integrated epithelium, consisting of undamaged enterocytes with numerous microvilli (Fig. 1). However, when the foregut was exposed to A. salmonicida, there were scattered areas of damaged epithelium, with de-epithelialised enterocytes observed in the foregut lumen (Fig. 2). Many of these enterocytes did not appear to be damaged, and had both intact microvilli and normal cytoplasm. Their shape had changed, however, to be more spherical compared with those enterocytes of the foregut not exposed to the pathogen. Moreover, in some locations, all the enterocytes had detached from the epithelium, thus exposing the basement membrane (results not shown). Normal microvillous architecture and undamaged enterocytes with numerous intracellular vacuoles were observed in the hindgut not exposed to pathogenic bacteria in the Ussing chamber (Fig. 3). In contrast, TEM examinations of the hindgut documented severe damage to microvilli (disintegrated), desmosomes and tight junctions (accompanied by increased intercellular spaces), in Fig. 2 TEM micrograph of the foregut of Atlantic salmon exposed to Aeromonas salmonicida ssp. salmonicida. Several detached or detaching enterocytes are seen in the gut lumen (arrows). ×3750. Bar: 7 μm enterocytes after mucosal exposure to A. salmonicida (Fig. 4). However, no de-epithelialised enterocytes were found in the hindgut lumen. No differences in the population level of adherent bacteria associated with the foregut (9×104) and hindgut (2×105) were found. A random selection of 24 strains isolated from the foregut and 25 isolates from the hindgut was classified into nine taxonomic groups, including Acinetobacter, Pseudomonas, Carnobacterium, Enterococcus, Staphylococcus, Micrococcus and Rhodococcus. Two taxonomic groups, Carnobacterium and Rhodococcus, were only isolated from the hindgut. Discussion Translocation of bacteria across the intestine, an essential and prerequisite step for bacterial invasion, cannot be effectively studied using in vivo models of translocation. In this respect, the Ussing chamber is a helpful tool to evaluate translocation of bacteria across the live intestinal epithelium of endothermic animals (Scheppach et al. 1996; Kurkchubasche et al. 1998; Isenmann et al. 2000; Mangell et al. 2002). Translocation of bacteria across the intestine in fish, defined as passage of viable bacteria from the digestive tract into enterocytes, has been reported in several investigations (for review, see Ringø et al. 2003). However, this phenomenon has mainly been observed for nonpathogenic indigenous gut bacteria, and not for pathogenic bacteria, and has not affected cellular integrity. The situation for fish pathogens seem to be completely 309 Fig. 3 TEM micrograph of the hindgut (posterior part of the intestine) of Atlantic salmon exposed to dextran containing Ringer (not exposed to bacteria) showing normal enterocytes with numerous vacuoles. L Lumen, MV microvilli, ER endoplasmic reticulum, G goblet cell, V vacuoles. ×3750. Bar: 5 μm different, as severe damage with loss of cellular integrity has been noted in the foregut of spotted wolffish (Anarhichas minor Olafsen) fry, infected by V. anguillarum (Ringø et al. 2003), as well as in vitro infection (present study, Fig. 2) and in vivo infection (Bakken 2002) of A. salmonicida ssp. salmonicida of Atlantic salmon. As no cell damage was observed in control fish (fish not exposed to pathogenic bacteria), we concluded that the indigenous bacteria isolated from foregut and hindgut in the present study do not affect cellular integrity. We observed detached, but almost intact, enterocytes in the foregut lumen (Fig. 2) after exposure to A. salmonicida in vitro. A similar result has also been reported in the pyloric caeca of Atlantic salmon in an in-vivo challenge experiment (Bakken 2002) and further by use of an in-vitro intestinal sack preparation (Ringø, Myklebust and Olsen unpublished data). However, in the in-vitro experiment of the present study, a quite different situation seems to occur in the hindgut region as no intact enterocytes were found in the lumen, but the microvilli were disintegrating and there was damage to intercellular tight junctions and desmosomes (Fig. 4). The differences between foregut and hindgut have not been elucidated, but it is probable that enterocytes in different regions of the GI tract vary in their susceptibility to pathogen-induced damage. This may be linked also to different regional rates of epithelial turnover 310 Fig. 4 TEM micrograph of the hindgut of Atlantic salmon exposed to A. salmonicida ssp. salmonicida shows severely damaged enterocytes. The microvilli (MV) are disintegrated. Increased intracellular gap (arrows) is seen in the apical region. L Lipid droplets, V vacuoles. ×3750. Bar: 5 μm or to different mechanisms of enterocyte loss by apoptosis or necrosis (Mayhew et al. 1999). Apoptosis-dependent processes tend to preserve junctional integrity whilst necrosis-like processes tend to be associated with junctional complex disruption and loss of microvillous morphology. In their recent review devoted to electron microscopy of the intestinal microbiota of fish, Ringø et al. (2003) suggested two different mechanisms to be involved in bacterial translocation of indigenous gut bacteria: (1) transcellular and (2) paracellular. However, the results of the present study by using the Ussing chamber method and the fish pathogen A. salmonicida clearly demonstrate the presence of intact enterocytes in the gut lumen (Fig. 2). This finding suggests a third mechanism of bacterial translocation, cell damage, a specific attack on tight junctions and desmosomes that leads to intact enterocytes in the lumen as a result of pathogen-mediated perturbation of intercellular junctions (loosening nexuses and desmosomes). However, in the present study, we were not able to demonstrate A. salmonicida in lamina propria and deeper sites of the intestinal tract. The reason for this has not been elucidated and is a topic for further studies. The difference in bacterial translocation between indigenous gut bacteria and pathogenic bacteria might be due to the production, by pathogens, of a great variety of virulence factors such as extracellular enzymes, outer surface components such as S-layer or secretory proteins, pore-forming toxins (Fivaz and van der Goot 1999). These factors could result in severe cell damage as demonstrated in foregut regions in the present study. It is well known from human studies that different bacteria species colonise different parts of the GI tract (Magigan et al. 2000), and that different pathogenic bacteria adhere to, and infect, different parts of the GI tract. Helicobacter pylori infect the gastric mucosa (Magigan et al. 2000), enterotoxigenic Escherichia coli the duodenum (Nataro and Kaper 1998) and enteroaggregative E. coli the jejunum, ileum or colon (Knutton et al. 1992; Hicks et al. 1996). Vibrio cholera infects the ileum (Bennish 1994), Salmonella the terminal ileum 311 (Finlay and Falkow 1989) and Shigella species the colon (Finlay and Falkow 1989). Based on our results, we suggest that the foregut of Atlantic salmon is an infection site in A. salmonicida ssp. salmonicida infection. However, as loosening of cell junctions was observed in the hindgut, this region is also probably involved in A. salmonicida infection but to a lesser extent than the foregut. When discussing intestinal cellular damages, the results of the present study obtained in the foregut (Fig. 2) are quite similar to the severe epithelial damage by intracellular fat accumulation by dietary linseed oil as observed by Olsen et al. (2000). In these studies, cell debris was also observed in the lumen giving free access to the basal membrane. References Bakken Y (2002) Histological studies of pyloric caeca of Atlantic salmon (Salmo salar L.) fed diets containing linseed, soybean, and marine oils. 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