Aquaculture 227 (2003) 395 – 415

www.elsevier.com/locate/aqua-online

Electron microscopy of the intestinal

microflora of fish
Einar Ringøa,*, Rolf Erik Olsenb,
Terry M. Mayhewc, Reidar Myklebustd
a
Department of Arctic Veterinary Medicine, The Norwegian School of Veterinary Science,
NO-9292 Tromsø, Norway
b
Institute of Marine Research, Matre Aquaculture Research Station, Matre, Norway
c
School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK
d
Institute of Anatomy and Cell Biology, University of Bergen, Bergen, Norway
Accepted 30 May 2003

Abstract

The role, and even the existence, of stable indigenous microbiota in fish was not accepted
until the 1970s. In the last decade, our understanding of adhesion and translocation of bacteria
in the fish gut has increased, and electron microscopy has contributed significantly to this
knowledge. This review summarises the information available on gut-associated bacteria and
on the translocation of bacteria in fish gastrointestinal tract.
In several studies on various fresh- and saltwater fish, bacteria in the intestinal lumen and
epithelium-associated bacteria have been demonstrated by using transmission electron
microscopy and/or scanning electron microscopy. Some of these studies have demonstrated
translocation of bacterial cells by endocytosis in the gastrointestinal tract of larvae and adult
fish as well as uptake of intact bacterial antigens. Endocytosis of bacteria in the digestive tract
is highly relevant as the gastrointestinal tract is a potential port of entry for pathogens.
D 2003 Published by Elsevier B.V.

Keywords: Electron microscopy; Digestive tract; Gut microbiota; Fish

1. Introduction

It is accepted that fish possess specific intestinal microbiota consisting of aerobic,

facultative anaerobic, and obligate anaerobic bacteria, but the bacterial composition
may

* Corresponding author. Fax: +47-77-69-49-11.

E-mail address: Einar.Ringo@veths.no (E. Ringø).

0044-8486/$ - see front matter D 2003 Published by Elsevier B.V.

doi:10.1016/j.aquaculture.2003.05.001
396 E. Ringø et al. / Aquaculture 227 (2003) 395–415

change with age, nutritional status, and environmental conditions. The intestinal
microbiota have been classified as autochthonous or indigenous (when they are able
to colonise the host’s gut epithelial surface) or as allochthonous or transient. In this
context, light and electron microscopic examinations of gut samples are important
tools for investigating the microbial ecology of the gastrointestinal (GI) tract
ecosystem and determining the presence of autochthonous or allochthonous
microbiota. Several studies on various fresh- and saltwater fish using transmission
electron microscopy (TEM) and/or scanning electron microscopy (SEM) have
demonstrated bacteria in the intestinal lumen and associated with the intestinal
epithelium (Lesel and Pointel, 1979; Austin and Al-Zahrani, 1988; Hansen and
Olafsen, 1999; Hansen et al., 1992; Kuperman and Kuz’mina, 1994; Grisez et al., 1996;
Bergh et al., 1997; Ringø and Olsen, 1999; Ringø et al., 2001, 2002; Hellberg and
Bjerkå s, 2000; Lødemel et al., 2001). Furthermore, some studies have revealed
translocation of bacterial cells by endocytosis in the GI tract of both fish larvae
(Hansen and Olafsen, 1999; Hansen et al., 1992; Grisez et al., 1996) and adult fish
(Ringø et al., 2001, 2002). Uptake of intact bacterial antigens has also been detected
(Hansen and Olafsen, 1990; Olafsen and Hansen, 1992).
This review provides an overview of electron microscopical studies on gut-
associated bacteria together with a critical evaluation of the results obtained so far.
This is highly relevant as the digestive tract is a potential port of entry for pathogens
(Chair et al., 1994; Olsson, 1995; Olsson et al., 1996; Grisez et al., 1996; Romalde et
al., 1996; Jö born et al., 1997; Robertson et al., 2000; Lødemel et al., 2001).
Prevention of disease requires knowl- edge about where the pathogens colonise
within the GI tract and in which regions the high- est frequencies of translocation
occur. Finally, directions for further research are proposed. As this review focuses on
electron microscopical studies on gut-associated bacteria in the fish intestine, readers
with special interest in bacterial species colonising the GI tract of different fish
species are referred to the comprehensive reviews on this topic by Cahill (1990),
Ringø et al. (1995), Hansen and Olafsen (1999) and Ringø and Birkbeck (1999).

2. The gastrointestinal tract

Many variations in morphology of the gastrointestinal (GI) tract exist between

various fish species. Depending on feeding habits and diet, it is generally accepted to
divide fish into carnivores (eating fish and bigger invertebrates), herbivores
(consuming mainly plant material), omnivores (mixed diet eaters) and detrivores
(feeding largely on detritus). Microphagous fish have less distensible tubes than
predatory fish.
The anatomy and physiology of the GI tract is covered elsewhere (Suyehiro,
1942; Fänge and Grove, 1979; Stevens, 1988; Olsen and Ringø, 1997). The digestive
system is essentially a muscular tract lined by a mucous membrane that exhibits
regional variations in structure reflecting the regional differences in function.

2.1. Stomach

This portion of the digestive tube possesses a distinctive cell lining, where acid is
secreted, usually along with some digestive enzymes like pepsin. However, not all
fishes
 E. Ringø et al. / Aquaculture 227 (2003) 395–415 397

have a stomach and it is lacking in lampreys, hagfish, chimaeras and in many

herbivorous fishes, e.g. minnows, carp (Cyprinidae), sauries (Scomberesocidae) and
parrotfishes (Scaridae). In most fish where a stomach is present, it may vary in
shape, size and structure according to diet and species (Suyehiro, 1942). The pH of
stomach in salmonid fish is between 3.0 and 4.5 (Ransom et al., 1984; Gislason et
al., 1996), and the bacterial
population level is in the range of 2 × 104 5
– 10 (Austin and Al-Zahrani, 1988;
Ringø,
1993). In electron microscopical studies, the stomach is rarely investigated.

2.2. Pyloric caeca

Unlike higher vertebrates, many fish have a number of pyloric caeca, from 1 to
1000, which are finger-like pouches that extend outward from the pylorus close to
where the intestine leaves the stomach. Pyloric caeca in fish serve to increase the
absorptive surface (Bergot et al., 1975). Some pyloric caeca are bifid (branched), but
the simplest ones resemble the human appendix. The number of pyloric caeca has
been used as one taxonomic criterion in identification of fish species. In addition, it
is worth noting that pyloric caeca are always absent in fish that lack a stomach. The
pH of caeca and caecal intestine is 7.0 and 7.5, respectively. Due to its complex
morphology, few microbial studies have investigated the microbiota of pyloric caeca
but the organ is normally included in electron microscopical investigations.
In pyloric caeca, the enterocytes are especially tall (between 50 and 80 Am) and
their microvilli are distinct. The enterocytes are polarised cells, possessing an apical
portion with few organelles (except for abundant small smooth-surfaced vesicles)
and a basal region containing mitochondria, abundant rough endoplasmic reticulum
and rough- surfaced vesicles. The nuclei, surrounded by profiles of the Golgi
complex, are located in the intermediate portion of the cell. Between the bases of
adjacent cells, paracellular spaces are commonly observed. One early investigation
reported that Vibrio ordalli were common in the pyloric caeca and located them just
beneath the intact mucosal epithelium (Ransom et al., 1984), while two recent
investigations clearly demonstrated bacteria associated to the epithelial surface of the
pyloric caeca (Ringø et al., 2001; Ringø, Olsen and Myklebust, unpublished data).

2.3. Intestine

The intestine is a complex multifunctional organ. In addition to digesting and

absorbing feedstuff, the intestine is critical for water and electrolyte balance,
endocrine regulation of digestion and metabolism, and immunity. The intestine has
many variations also in both length and arrangement (Fänge and Grove, 1979;
Stevens, 1988), and its structural diversity in fish (Suyehiro, 1942) exceeds that
found in other vertebrates (Stevens, 1988). The posterior part of the intestine is
considered to be the main site for intestinal absorption of macromolecules in
salmonids and in some other fish species (for review, see Dalmo et al., 1997). The
midgut begins immediately posterior to the pylorus and has distinct epithelial,
absorptive and secretory cells. The hindgut is an extension of the midgut with
gradually diminishing digestive and absorptive functions, and increased levels of
mucus production. Intestinal pH is between 7.0 to 8.0 and 7.5 to 9.0 in the small and
large
398 E. Ringø et al. / Aquaculture 227 (2003) 395–415

intestine, respectively (Ransom et al., 1984; Gislason et al., 1996). The gut
epithelium of both midgut and hindgut has extensive foldings. The bacterial
population level of the intestines has been investigated in several studies (for review,
see Cahill, 1990; Ringø et al., 1995), and most of these studies have shown a
progressive increase in number of aerobic heterotrophic bacteria associated with the
intestine from about 105 in small intestine to 107 in large intestine of adult fish
species.
Proliferation of stem cells is much slower in fish relative to mammals, even when
temperature is considered. This may be a result of slower rates of DNA synthesis
(Danguy et al., 1988). As a result, cell replacement rates are much lower in fish and
enterocytes can remain on the villi for weeks (e.g., 15 – 20 days for grass carp,
Ctenopharyngodon idella; Stroband and Debets, 1978). This contrasts with the 2 – 3-day
lifespan characteristic of mammalian enterocytes (see Mayhew, 1990).
The morphology of enterocytes in small intestine is similar to that found in the
pyloric caeca. However, the enterocytes in large intestine are somewhat different, as
they are generally shorter, but of approximately the same width. There may be
extensive endocytic activity at the bases of microvilli and many of the endocytic
vesicles contain electron- dense material, and there are the numerous
intracytoplasmic filament bundles. In the mid- portion of enterocytes, there are
numerous relatively large vacuoles (0.5 – 1 Am in diameter) containing material of
variable electron-density.

3. Scanning electron microscopy

Several factors influence adhesion and colonisation of the microbiota within the
digestive tract. These include: (1) gastric acidity, (2) bile salts, (3) peristalsis, (4)
digestive enzymes, (5) immune response and (6) indigenous bacteria and the
antibacterial com- pounds which they produce. Bacterial adhesion is a cell-surface
interaction phenomenon and this property makes it ideal for examination by scanning
electron microscopy (SEM) (Knutton, 1995). Compared to transmission electron
microscopy (TEM), SEM has the advantage that large areas of cell surface can be
examined rapidly for adherent bacteria. Lesel and Pointel (1979) used SEM and
classical microbiology to investigate bacteria in various regions (stomach, pyloric
caeca, anterior part of intestine and middle portion of intestine) of the digestive tract
of rainbow trout (Oncorhynchus mykiss Walbaum). While the microflora present in
stomach and intestine determined by classical microbiology seems to be identical in
numbers, with SEM it was only possible to observe bacteria associated with pyloric
caeca (bacilliform organisms) and middle intestines. However, the authors discovered
that some differences occurred with respect to bacteria colonising the two regions as
bacilliform organisms were found in the pyloric caeca, while two morphologically
different forms [(1) long rods, rectilinear, with very slim extremities, and (2)
undulated, spiralled with sharp-pointed extremities] were observed in middle
intestines.
Later, Austin and Al-Zahrani (1988) showed some bacteria-like objects
sporadically associated with the stomach wall of rainbow trout, and estimated that the
average coverage of the wall with such entities was < 0.001% of the total area.
However, many more organisms, of a coccoid morphology, were observed in
association with food particles in
 E. Ringø et al. / Aquaculture 227 (2003) 395–415 399

the stomach. Moreover, the authors stated that microscopy failed to produce any
evidence for the presence of yeast, mycelial fungi, diatoms or protozoa within the fish
GI tract.
Magarinos et al. (1996) demonstrated that Pasteurella piscicida strains adhered
strongly to the intestines from sea bream, sea bass and turbot in numbers ranging
from 104 to 105 bacteria per gram of intestine depending on the bacterial isolate and
the fish species employed. These results were clearly supported by the scanning
electron microscopy studies.
In a recent investigation, Ringø and Olsen (1999) raised the question of whether
or not there is a connection between SEM investigations and the dilution plate
technique when determining bacteria associated with the small and large intestine of
Arctic charr (Salvelinus alpinus L.) fed diets containing high (24%) and low (6%)
levels of carbohydrate. The authors reported a predominance of cocci determined by
classical microbiology, a finding confirmed by SEM of the enterocytes (Fig. 1).
More recently, Ringø et al. (2001) showed substantial associations of both
coccoid and rod-shaped bacterial cells with the apices of, and between, microvilli of
the enterocytes in the midgut of Arctic charr fed soybean oil (Fig. 2). As seen in Fig.
2, some enterocytes were heavily colonised while others showed no associated
bacteria. The reason for this difference in colonisation of the enterocyte surface was
not elucidated, but Ringø et al. (2001) suggested three possible causes; (1) enterocyte
ageing, (2) differentiation, or (3) specific receptors for receptor-mediated
endocytosis of bacteria. Clear differences in bacterial colonisation of the enterocyte
surface in the hindgut were also observed when Arctic charr were fed dietary
soybean oil or linseed oil (Ringø et al., 2002). Some enterocytes were heavily
colonised by bacteria when the charr were fed soybean oil (Fig.

Fig. 1. Scanning electron microscopy (SEM) micrograph showing coccoid-shaped bacteria associated with
enterocytes in the hindgut of Arctic charr (S. alpinus L.). × 10000. After Ringø and Olsen (1999).
400 E. Ringø et al. / Aquaculture 227 (2003) 395–415

Fig. 2. SEM micrograph of the apical aspects of enterocytes in the midgut of Arctic charr fed soybean oil.
The borders between adjacent cells are clearly visible as are the microvilli which cover the cell apex. The
luminal ends of bacteria located in the interstices between microvilli are also visible (arrows). × 7500.
After Ringø et al. (2001).

Fig. 3. SEM micrograph showing bacteria associated with enterocytes in the hindgut of Arctic charr fed
linseed oil. Associated bacteria (arrows) are located at the apical brush border. × 10000. After Ringø et al.
(2002).
 E. Ringø et al. / Aquaculture 227 (2003) 395–415 401

2), while in fish fed dietary linseed oil, most bacteria associated with enterocytes
were located at the apical brush border (Fig. 3). These differences in bacterial
colonisation of enterocyte surface between fish fed different vegetable oils may be
related to the different gut microbiota observed between the two dietary groups
(Ringø et al., 2002). This controversial hypothesis calls for further investigations.

4. Transmission electron microscopy

TEM of sectioned material, with or without immunolabeling, has been used

primarily to provide high-resolution information about the mechanisms of interaction
between bacteria and the enterocyte cell surface. Numerous reports published in the
1970s and 1980s used classical microbiology methods to describe bacteria associated
with the intestine, but it was not until the last decade that TEM investigations
confirmed these results. TEM studies on the digestive tract of herring (Clupea
harengus L.) larvae at the yolk sac stage confirmed the classical microbiology and
light microscopic findings with respect to distribution of bacteria along the GI tract
(Hansen et al., 1992).
In an ultrastructural study on the intestinal epithelium in three species of
freshwater fish (pike, Esox lucius L.; burbot, Lota lota L.; bream, Abramis brama
L.), Kuperman and Kuz’mina (1994) detected only coccoid-shaped bacteria
associated with the enterocyte microvilli of the caudal segment of the posterior part
of pike intestine, but bacteria were seen in only small amounts.
In their study, devoted to clarifying the infection route of the fish pathogen Vibrio
anguillarum in turbot (Scophthalmus maximus L.) larvae after oral challenge through
live feed, Grisez et al. (1996) showed positively stained bacteria in the intestinal
lumen, attached to the brush border of the epithelium of the anterior part of the
intestine and occasionally in epithelial cells.
In a later study, the susceptibility of early life stages of turbot and Atlantic
halibut (Hippoglossus hippoglossus L.) to Aeromonas salmonicida subsp. salmonicida
was studied in challenge experiments (Bergh et al., 1997). Larvae of both species
experienced high mortality during the yolk sac stage, and the authors suggested that
this was as a result of the challenge test. However, the bacterium could not be
recovered from the larvae by culture, but the pathogen was shown to be present in
the intestinal lumen of some turbot larvae examined using immunohistochemical
techniques. Based on this result, the authors (Bergh et al., 1997) proposed that Aer.
salmonicida subsp. salmonicida may persist in the larvae. More attention has been
paid to the importance of TEM investigations in larval rearing, and in a recent study,
Hansen and Olafsen (1999) demonstrated rod-shaped bacteria associated with the
tips of microvilli in the anterior part of the hindgut in a 14-day-old herring
(Clupea harengus L.) larva incubated in sand-filtered seawater (Fig. 4). Numerous
rod-shaped bacteria with a granular cytoplasm and distinct cell wall have also
been observed between the microvilli in the distal intestine of common wolffish
(Anarhichas
lupus L.) (Fig. 5) (Hellberg and Bjerkå s, 2000).
One remarkable feature of the indigenous intestinal microbiota of fish is that they
are affected by certain situations including stress, antibiotic administration and even
small dietary changes. The stability of the intestinal flora is an extremely important
factor in the
402 E. Ringø et al. / Aquaculture 227 (2003) 395–415

Fig. 4. Bacteria associated with microvilli in the anterior part of the hindgut in a 14-day-old herring larva
incubated in sand-filtered seawater. Larvae were washed in sterile filtered seawater. Bar = 1.0 Am. After
Hansen and Olafsen (1999).

Fig. 5. Ultramicrograph of distal intestine showing the apical cytoplasm and microvilli (MV) of the
columnar epithelial cells of common wolffish (A. lupus L.). The 1.5-Am-long microvilli are covered by
mucus (GM) from goblet cells. A bacterium (arrow) is seen oriented in parallel with the microvilli.
Desmosome (D). × 30000. Inset: Transverse section of brush border showing microvilli and bacteria
(arrows). × 30000. After Hellberg and Bjerkås (2000).
 E. Ringø et al. / Aquaculture 227 (2003) 395–415 403

natural resistance of fish to infections produced by bacterial pathogens in the

intestinal tract. The emergence of hierarchy formation and excessive handling stress
affect the GI microbiota (Ringø et al., 1997; Ringø, Olsen and Myklebust,
unpublished results). In a recent study, Ringø, Olsen and Myklebust (unpublished
results) clearly demonstrated

Fig. 6. TEM micrograph of Atlantic salmon gut epithelium (pyloric caeca). Several enterocytes are seen
with distinct brush-border (MV). In the gut lumen (GL) profiles of rod-like bacteria (B) are seen. Note the
penetration of a rod like bacterium into the cytoplasm of an enterocyte (arrow). Note also that there seems
to be a membrane reaction at the penetration site (dark material) (arrowhead). After Bakken, Myklebust,
Olsen and Ringø (unpublished results).
404 E. Ringø et al. / Aquaculture 227 (2003) 395–415

substantial numbers of rod-shaped bacteria associated with the microvilli of

enterocytes in the midgut of Atlantic salmon (Salmo salar L.), even 4 h after
extensive handling stress. This increase may be a consequence of the peel-off of
mucus which then allows bacteria to colonise the area between the microvilli. Again,
testing this controversial hypothesis will require further investigations.
The mucous blanket is constantly renewed by the secretion of high molecular
weight glycoproteins from individual goblet (mucus producing) cells throughout the
epithelium. Goblet cells differentiate in the lower portion of the crypts of both small
and large intestine and gradually migrate onto the villi or mucosal surface. In an
early study on histopathol- ogy changes caused by V. anguillarum, Ransom et al.
(1984) found large amounts of goblet cells in the anterior part of GI tract of infected
chum salmon (Oncorhynchus keta). In a later study, it was clearly demonstrated that
the first reaction of Arctic charr infected by pathogenic bacteria (Aer. salmonicida) is
to peel off the infected mucus by increased goblet cell production (Lødemel et al.,
2001). A similar reaction to that found in infected fish is also observed in rabbit and
rats infected by pathogenic bacteria (Mantle et al., 1989, 1991; Enss et al., 1966).
Certain components of the resident bacteria of the intestine are responsible for
enteric bacterial antagonism and colonization resistance, since they are associated
closely with the intestinal epithelium and form a barrier serving as the first defence
to limit direct attachment or interaction of pathogenic bacteria to the mucosa. Both
pathogenic and normal enteric microflora can induce and perpetuate chronic
intestinal inflammation with systemic manifestations in genetically susceptible hosts
(Sartor, 1997). Studies on endothermic animals have demonstrated that not all
luminal bacteria have equal capacities to induce mucosal injury, since some species
can induce inflammation (Bacteroides), some are neutral (Escherichia coli) and
others may be protective (lactoba- cilli). Inflammation of the gut enterocytes by
penetration of intestinal bacteria has not been investigated in fish, but in a recent
study it was clearly demonstrated by TEM that inflammation does occur in fish
intestinal enterocytes when a rod-like bacterium was observed apparently penetrating
into the cytoplasm of an enterocyte (Fig. 6). A clear inflammation reaction is seen at
the penetration site.

5. Bacterial translocation

In mammals, bacterial translocation from the GI tract is a well-known

phenomenon, and is defined as the passage of viable bacteria from the digestive tract
to extraintestinal sites such as liver, spleen or bloodstream (Berg, 1995).
Translocation of bacteria is favoured when there is intestinal bacterial overgrowth,
deficiencies in the host immune defence system or damage to the intestinal mucosal
barrier (Berg, 1992). Indigenous intestinal flora are prevented from gaining access to
other sites in the body by a single epithelial layer in the mucosa. Intestinal mucins,
secreted by specialised epithelial goblet cells located in the intestinal epithelium,
form a viscous, hydrated blanket on the surface of the mucosa that protects the
delicate columnar epithelium. This mucous coat is thought to be a vital component of
the intestinal mucosal barrier in preventing microbial colonisation of pathogens in
both fish and endothermic animals (Westerdahl et al., 1991; Maxson et al., 1994;
Mims et al., 1995). There are thought to be three major functions of the GI mucus;
 E. Ringø et al. / Aquaculture 227 (2003) 395–415 405

(1) protection of the underlying mucosa from chemical and physical damage, (2)
lubrication of the mucosal surface, and (3) providing a barrier against adherence of
pathogenic organisms to the underlying mucosal epithelium.
The pathogenesis of vibrio infections in mammals is primarily a gut infection, and
it is therefore logical to ask whether the same is true in fish. Fish pathogenic
bacteria, such as Vibrio salmonicida and V. anguillarum, have been shown in vivo to
adhere to the intestinal epithelium of fish larvae and to promote severe destruction of
microvilli (Olafsen and Hansen, unpublished results). Severe damage with loss of
cellular integrity was also noted in the midgut of spotted wolffish (Anarhichas minor
Olafsen) infected by V. anguillarum (Ringø, Mikkelsen and Myklebust, unpublished
data) (Fig. 7). SEM investigations of human intestinal mucosa infected with
enteropathogenic E. coli (EPEC), showed that EPEC adhere intimately in
microcolonies and cause gross alterations at the apical surface of infected
enterocytes (Knutton et al., 1987; Knutton, 1995).
Endocytosis of intact bacterial antigens and translocation of bacterial cells by
endocy- tosis have been reported in the GI tract of fish larvae (Hansen and Olafsen,
1990, 1999; Hansen et al., 1992; Olafsen and Hansen, 1992; Grisez et al., 1996). In
the family Gadidae, the gut is clearly segmented 6-day post hatching. Consequently,
gross morphological

Fig. 7. Spotted wolffish (A. minor Olafsen) fry infected by V. anguillarum. Notice the severe cellular
damage of enterocytes (Ringø, Mikkelsen, Kaino and Myklebust, unpublished results).
406 E. Ringø et al. / Aquaculture 227 (2003) 395–415

changes affect the GI tract of Atlantic cod (Gadus morhua L.) larvae before food uptake
starts. However, 4-day-old yolk sac larvae of Atlantic cod ingest bacteria by drinking
and by swallowing mucus with bacteria (Olafsen, 1984). Hansen and Olafsen (1990)
reported that endocytosis of intact bacterial antigen occurred in a restricted area of the
foregut (near the oesophagus) of 4-day-old cod larvae. Vibrio fischeri, the fish pathogen
V. salmonicida, and a Flavobacterium sp. were all engulfed by endocytotic cells, but the
uptake varied between bacterial species in the order V. fischeri>V.
salmonicida>Flavobacterium sp.
Thus, bacteria enter the digestive tract before the gut is developed and active
feeding commences. At this developmental stage, the level of digestive enzymes in
the gut is low (Hjelmeland et al., 1988), and probably does not affect the bacterial
surface ligands to the same extent as in older fish. TEM investigations have
demonstrated the uptake of intact antigen in both Atlantic cod and herring (Olafsen
and Hansen, 1992).
Hansen et al. (1992) investigated the digestive tract of herring larvae and
suggested that endocytosis occurs in the posterior part of the gut, but it was difficult to
display endocytosis convincingly. However, the authors demonstrated ciliated cells
among the epithelial cells and suggested that ciliated cells may take part in cleansing
bacteria from the brush border and/or in effectively transporting them towards the
posterior part of the gut of marine fish larvae. Endocytosis of bacteria by
enterocytes was however observed in the hindgut of herring larvae (Hansen and
Olafsen, 1999) (Fig. 8). In their study on turbot larvae, Grisez et al. (1996) reported
free V. anguillarum from an endosome in the lamina propria by
immunohistochemical staining (Fig. 9). Spotted wolffish is another marine fish
species with considerable potential for aquaculture because it hatches as fry and has
an excellent meat quality. In a recent study (Ringø et al., unpublished results),
endocytosis of several bacteria was demonstrated by enterocytes in the hindgut of
spotted wolffish fry (Fig. 10). In their study on intestinal uptake of V. anguillarum
bacterin in juvenile rainbow trout, sea bass and turbot, Vigneulle and Baudin
Laurencin (1991) used oral administration and anal intubation. Antigen, detected on
the basis of its specific fluorescence was observed in the posterior intestine. These
results were later confirmed by Tamura et al. (1993) in adult eel (Anguilla anguilla).
Vigneulle and Baudin Laurencin (1991) also noted some difference between the
two delivery methods, as fluorescence was always more pro- nounced after anal
intubations, regardless of the fish species. Some differences in bacterin uptake were
apparent between the fish species, as the intestinal uptake seemed to be less
efficient in rainbow trout than in turbot and sea bass. In order to understand some
of the possible mechanisms involved in translocation of V. anguillarum in juvenile
turbot, Olsson et al. (1996) used TEM. However, in vitro examination showed that V.
anguillarum cells did not adhere specifically to intestinal mucus but, instead,
accumulated close to rectal
epithelium. No epithelial cell penetration or endocytosis was evident.
When discussing endocytosis, it is important to take account of the ontogeny of
the digestive tract of fish (this differs morphologically and functionally between
taxa) and the development of the digestive tract. At the time of hatching, the digestive
tract of teleosts is an undifferentiated straight tube which is morphologically and
physiologically less elaborate than that of the adult. However, it differentiates into a
histologically and functionally distinct fore-, mid-, and hindgut at about the time of
first exogenous feeding (Govoni et al., 1986). Based on the gradual development,
one might raise the question of whether or not endocytosis occurs in adult fish.
Endocytosis of macromolecules has been
 E. Ringø et al. / Aquaculture 227 (2003) 395–415 407

Fig. 8. Endocytosis of bacteria demonstrated in enterocytes in the posterior part of the hindgut in a 14-day-
old herring larva. Bar = 1.0 Am. After Hansen and Olafsen (1999).

observed in a number of juvenile and adult fish species as well as endocytosis of

bacteria cells in the pyloric caeca (Fig. 11), midgut and hindgut of adult Arctic charr
(Ringø et al., 2001, 2002).
In bacterial translocation from the GI tract, two different mechanisms can be
involved;
(1) intracellular and (2) paracellular. In intracellular translocation, two different
routes are involved; (a) endocytosis (Fig. 10) and (b) penetration (Fig. 6). It is well
accepted that the cell junctions consist of two different regions; (i) tight junctions
and (ii) a desmosome area. Paracellular translocation must involve loosening of cell
junctions. Loosening of cell junctions has however not been observed, while
loosening of desmosomes has been observed. The intracellular proliferation of
bacteria involves two different mechanisms; (a)
408 E. Ringø et al. / Aquaculture 227 (2003) 395–415

Fig. 9. V. anguillarum infection in turbot after oral challenge. Immunohistochemical staining. Release of V.
anguillarum from an endosome into the lamina propria (arrow). After Grisez et al. (1996).

endocytosis of bacteria into cytoplasm, and it is transported to the lamina propria

without any damage or bacteria may lyse as a result of lysosomatic activity and (b)
bacteria are encapsulated by a membrane vacuole which could protect the bacteria
against enzymes and lysosomes. Bacterial encapsulation by membrane vacuoles is
observed into tonsillar surface epithelium during infectious mononucleosis (Stenfors
et al., 2000). Furthermore, the pathogenic bacteria have to penetrate the basal
membrane. Mechanisms involved in this have not been elucidated, but may involve
enzymatic activity. The mechanisms involved in bacterial translocation must be
investigated in greater details in order to evaluate differences between the indigenous
microflora and pathogenic bacteria, and possible differences involved in
translocation between different pathogens. This is a topic for further studies.
From a microbial point of view, it is important to have stable intestinal microbiota
as a part of the natural resistance of fish to infections. Therefore, a fundamental
question is: What happens to the indigenous intestinal microbiota during infection?
In a recent investigation, Lødemel et al. (2001) showed substantial numbers of
bacteria associated with the microvilli of Arctic charr prior to challenge with Aer.
salmonicida subsp.
 E. Ringø et al. / Aquaculture 227 (2003) 395–415 409

Fig. 10. TEM of enterocytes in the hindgut of spotted wolf fish fry. Several bacterial profiles are seen
(arrows).
× 3000. After Ringø, Mikkelsen, Kaino and Myklebust (unpublished results).

salmonicida. However, few bacteria were found associated with the microvilli of
infected fish. These results are consistent with those of Ringø et al. (2002) who
demonstrated an approximately 100-fold lower population level of bacteria
associated with the digestive tract of infected fish. This lower level of bacteria in
infected fish may be due to loss of mucus during infection, and implies a loss of the
protective microbiota which may prevent adherence and colonisation of pathogenic
bacteria.
Magarinos et al. (1996) evaluated the invasive capacities of the fish pathogen P.
piscicida, and the results on the invasion of different poikilothermic cell lines by the
pathogen indicated that following the Janda index (Janda et al., 1991), the P.
piscicida strains can be considered weakly or moderate invasive, with the number of
intracellular bacteria ranging from 104 to 105 per gram. P. piscicida was able to
invade the cell line CHSE-214 and to remain viable inside the infected cells at least
for 2 days.
From studies on endothermic animals, it is clear that specialised structures of the
intestinal epithelium may allow natural entry of bacterial pathogens. The interaction
of bacteria with a specialised epithelial cell, the M cell, requires adhesion as a
prelude to crossing the epithelial barrier (Jones et al., 1995; Neutra et al., 1996;
Vazquez-Torres and Fang, 2000). Unfortunately, information about the interactions
between intracellular
410 E. Ringø et al. / Aquaculture 227 (2003) 395–415

Fig. 11. TEM field of the apical regions of enterocytes in the pyloric caeca of Arctic charr. Bacterial
profiles are seen scattered at different levels within the brush border from the tips to the bases of
microvilli. In addition, one bacterial profile (arrowhead) is seen to be bound in an internalised, membrane-
bound endocytic vacuole.
× 15000. After Ringø et al. (2001).

pathogenic bacteria and M cells in fish is not available and this should be a topic of
future studies.

6. Immunogold labelling

Immunocytochemical techniques are powerful tools for localising specific

antigens, but the method is an underutilised technique to investigate the presence,
distribution and relative abundance of bacterial fish pathogens.
 E. Ringø et al. / Aquaculture 227 (2003) 395–415 413

Bacterial translocation from the gut is a complex process whereby indigenous

intestinal microflora relocate from the lumen (Finlay and Falkow, 1997) and is an
important phenomenon in the pathogenesis of ‘‘opportunistic’’ infections by
indigenous intestinal bacteria. Once inside a host cell, pathogens have a limited
number of ways to ensure their survival whether remaining within a host vacuole or
escaping into the cytoplasm. Avoidance of the host immune defences is a key to the
success of a pathogen. Several common themes are employed, including antigenic
variation, camouflage by binding host molecules, and enzymatic degradation of host
immune components.
Controversy also exists as to whether the intestine is an infection route for Aer.
salmonicida. Hiney et al. (1994) located Aer. salmonicida in the intestine of Atlantic
salmon (Salmo salar L.) by enzyme-linked immunosorbent assay and suggested that
the intestine might be the primary location of Aer. salmonicida with stress-induced
infections. In contrast, Svendsen et al. (1999) suggested that the skin of Atlantic
salmon is the major

Fig. 12. Immunogold-labelled Aer. salmonicida subsp. salmonicida from the midgut of Arctic charr fed
marine oil diet. Gold particles are marked with arrows. × 7500. After Lødemel et al. (2001).
412 E. Ringø et al. / Aquaculture 227 (2003) 395–415

infection route of the pathogen. However, in a recent study, Lødemel et al. (2001)
detected
Aer. salmonicida within midgut enterocytes of Arctic charr (Fig. 12).

7. Future perspectives

It is well known that the pathogenesis of Vibrio infections in mammals is

primarily a gut infection, and it is therefore logical to ask whether the same is true of
pathogenic Vibrio and Aeromonas infections in fish. As the GI tract seems to be
involved in pathogenic infections in fish (Chair et al., 1994; Olsson, 1995; Olsson et
al., 1996; Grisez et al., 1996; Romalde et al., 1996; Jö born et al., 1997; Robertson et
al., 2000; Lødemel et al., 2001), an important question in microbial ecology is how to
defeat pathogens in the GI tract? We suggest a new strategy: the microbiologist must
think as the military and use military strategy. If we are going to defeat our enemies,
the pathogens, we have to be at the same place and time as them. This important point
can explain why most probiotics studies on fish do not have any clear effect in
disease studies. For example, if probiotic bacteria mostly colonise the pyloric caeca,
the probionts will have no effect if the pathogen mostly colonises the mid or hindgut
regions and translocate in these regions. In this respect, electron microscopical
studies may be a useful tool in evaluating which part of the GI tract the probionts
colonise and where the main translocation of the pathogen occurs.
Another aspect on fish health is modulation of immune response and barrier
function by dietary means. It is well known from endothermic animals that any
method that leads to an improved feed intake in the first days after weaning could
reduce inflammatory symptoms, improve intestinal morphology and resistance
against pathogens (Bosi, 2000). However, knowledge about dietary tools to modulate
the immune response and barrier function in the fish gut is incompletely known, and
this is a topic of further study.

Acknowledgements

We are indebted to Ms. Turid Kaino for her excellent technical assistance. The
authors are grateful to Drs. Grisez and Hansen, Mr. Lødemel, Ms. Hellberg and Ms.
Bakken for providing their photographs. Financial support from Norwegian Council
(grant no. 122851/122) is gratefully acknowledged.

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