Download Damaging effect of the fish pathogen Aeromonas salmonicida ssp

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.
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