Experimental infection of Flavobacterium psychrophilum in fins of Atlantic salmon Salmo salar revealed by scanning electron microscopy

Abstract

Infections caused by Flavobacterium psychrophilum include 'bacterial coldwater disease' (BCWD) and 'rainbow trout fry syndrome' (RTFS), which are severe diseases that can cause high mortality and significant losses in hatchery-reared salmonids worldwide. Usually, these conditions start with necrosis along the edge of the fins. As the infection progresses, both the fish surface and the internal organs can be involved. The aetiological agent produces a Ca-dependent protease that can be responsible for some of the pathogenic responses, although the precise nature of the response remains to be elucidated. Atlantic salmon Salmo salar were experimentally infected by F. psychrophilum in order to investigate the bacterial invasion in the fin tissues by scanning electron microscopy. The images showed numerous bacteria embedded in the mucous layer when this remained on the tegument. In other zones without mucus, it was observed that bacteria were present on the axis of fin rays, but not on the epidermal surface. The material on these axes was largely eroded by tubular boreholes, and bacterial rods could be seen in these perforations. EDX (Energy Dispersive X-ray) microanalysis of the axis of the fin rays showed significant amounts of P and Ca, revealing the ossification of the ray axis. The protease activity could explain the formation of the tubular boreholes, allowing the bacteria the necessary Ca for the activation of the enzyme. The erosion pattern suggests that the gliding motility of F. psychrophilum could be involved in this burrowing ability.

Full text

DISEASES OF AQUATIC ORGANISMS
Dis Aquat Org
Vol. 59: 79– 84, 2004 Published April 21
INTRODUCTION
Flavobacterium psychrophilum (formerly Cytophaga
psychrophila and Flexibacter psychrophilus) (Bernardet
et al. 1996) is the aetiological agent of the diseases
referred to as ‘bacterial cold water disease’ (BCWD)
(Wood & Yasutake 1956) and ‘rainbow trout fry syn-
drome’ (RTFS) (Austin & Stobie 1991), also called ‘fry
mortality syndrome’ (Lorenzen et al. 1991) and ‘rain-
bow trout fry anaemia’ (RTFA) (Bruno & Poppe 1996).
These are septicaemic infections that are particularly
serious in juvenile fish that are not fully immunocom-
petent. Severe outbreaks with significant early losses
have been reported in hatchery-reared salmonids
world-wide, particularly rainbow trout Oncorhynchus
mykiss in Europe and coho salmon O. kisutch in Amer-
ica, but several non-salmonid fish species have also
been affected (Dalsgaard 1993, Nematollahi et al.
2003a). Eyed (Rangdale et al. 1997) and unfertilised
eggs (Vatsos et al. 2001) likewise were infected.
Currently, F. psychrophilum is well characterised by
different genetic and molecular techniques that differ-
© Inter-Research 2004 · www.int-res.com
*Email: juanlm@uniovi.es
NOTE
Experimental infection of Flavobacterium
psychrophilum in fins of Atlantic salmon Salmo
salar revealed by scanning electron microscopy
Juan Luis Martínez1,*, Alín Casado2, Ricardo Enríquez2
1
Departamento de Biología de Organismos y Sistemas, Universidad de Oviedo, c/ Catedrático Rodrigo Uría s/n, 33071 Oviedo, Spain
2
Laboratorio de Ictiopatología, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile
ABSTRACT: Infections caused by Flavobacterium psychrophilum include ‘bacterial coldwater dis-
ease’ (BCWD) and ‘rainbow trout fry syndrome’ (RTFS), which are severe diseases that can cause
high mortality and significant losses in hatchery-reared salmonids worldwide. Usually, these condi-
tions start with necrosis along the edge of the fins. As the infection progresses, both the fish surface
and the internal organs can be involved. The aetiological agent produces a Ca-dependent protease
that can be responsible for some of the pathogenic responses, although the precise nature of the
response remains to be elucidated. Atlantic salmon Salmo salar were experimentally infected by F.
psychrophilum in order to investigate the bacterial invasion in the fin tissues by scanning electron
microscopy. The images showed numerous bacteria embedded in the mucous layer when this
remained on the tegument. In other zones without mucus, it was observed that bacteria were present
on the axis of fin rays, but not on the epidermal surface. The material on these axes was largely
eroded by tubular boreholes, and bacterial rods could be seen in these perforations. EDX (Energy
Dispersive X-ray) microanalysis of the axis of the fin rays showed significant amounts of P and Ca,
revealing the ossification of the ray axis. The protease activity could explain the formation of the
tubular boreholes, allowing the bacteria the necessary Ca for the activation of the enzyme. The ero-
sion pattern suggests that the gliding motility of F. psychrophilum could be involved in this
burrowing ability.
KEY WORDS: Flavobacterium psychrophilum · Scanning electron microscopy · Salmonid · Fin rays ·
Pathogenesis · Bacterial coldwater disease (BCWD) · Rainbow trout fry syndrome (RTFS)
Resale or republication not permitted without written consent of the publisher

Dis Aquat Org 59: 79–84, 2004
entiate this species from other closely related bacteria
found in diseased salmonid fish (Crump et al. 2001,
Bader & Starliper 2002, del Cerro et al. 2002).
Although Flavobacterium psychrophilum is respon-
sible for a wide range of conditions of the external (and
occasionally internal) tissues of the fish, early signs
mainly affect the fins as a line of whitish material along
the margin. Fin rays may also begin to separate, and
the disease progresses inwardly on the fins until the
base of the attachment of the fins is reached (Shotts &
Starliper 1999, Bader & Starliper 2002).
Characteristics of Flavobacterium psychrophilum
have caused difficulties in challenge methods with
the bacteria (Decostere et al. 2000), but successful
attempts at experimentally inducing the disease have
been reported, and several methods have been
described, such as intraperitoneal, subcutaneous or
intramuscular injections, baths, and patches on the
skin (Madsen & Dalsgaard 1999, Garcia et al. 2000,
Ekman 2003). The precise nature of the pathogenic
mechanism of F. psychrophilum is poorly understood,
but since Pacha (1968) postulated that the proteolytic
nature of the bacteria plays a part in the mode of
pathogenesis, an important role for extracellular pro-
teases produced by the bacteria has been recognised
(Otis 1984, Madsen & Dalsgaard 1998, Crump et al.
2001, Secades et al. 2001).
Kondo et al. (2002) showed the adherence of Flavo-
bacterium psychrophilum on the surface of the ayu
Plecoglossus altivelis, and Rangdale et al. (1999)
histopathologically and ultrastructurally examined the
spleen in cases of rainbow trout fry syndrome, but no
electron microscopical study was made to analyse the
features of the F. psychrophilum infection in salmonid
fins. In this study, Atlantic salmon Salmo salar were
experimental infected by F. psychrophilum, and their
fins observed by scanning electron microscopy (SEM)
in order to investigate the invasion of bacteria in the
tissue.
MATERIALS AND METHODS
Bacterial cultivation and preparation. Flavobacterium
psychrophilum Strain R.128 was originally isolated
at the Ichthyopathology Laboratory (Universidad Aus-
tral de Chile) from diseased fish displaying characteris-
tic signs of RTFS. Bacteria were grown in modified
Anacker and Ordal agar (MAOA) (0.5% typtone,
0.05% yeast extract, 0.02 % sodium acetate, 0.02% beef
extract, 1.5% agar) (Lorenzen et al. 1997) at 15°C for 3 d
and washed twice in PBS pH 7.2 centrifuged at 4000
rpm (2021 ×g) for 20 min. The bacterial solution pre-
pared for experimental infection was 3.3 ×108colony
forming units (CFU) ml–1 in a total volume of 30 ml.
Fish and experimental infection. Juvenile Atlantic
salmon Salmo salar, n = 30, initial mean weight 17.3 g,
were obtained from a hatchery, transferred to the
Ichthyopathology Laboratory and controlled in accor-
dance with the O.I.E procedures (OIE 1997). Fish were
randomly separated into 2 groups. Subsequently, one
group was immersed for 1 h in an aquarium with 3 l of
freshwater at 15°C containing Flavobacterium psy-
chrophilum at a final concentration of 3.3 ×106CFU
ml–1. The other group, control fish, was introduced to a
similar aquarium with 3 l of freshwater at 15°C (in the
absence of bacteria). Each group was afterwards
moved to 80 l freshwater aquariums.
Sampling procedure. In the exposed group, samples
were obtained at 0, 24, and 48 h post infection times
(PIT). Three fish of each PIT group were sacrificed and
the dorsal fins were removed. In one sample of each
PIT group, the tips of the fin rays were cut off with a
scalpel. Likewise, samples were obtained from control
fish. The samples were fixed in Karnowsky fixative
(2.5% glutaraldehyde, 4% paraformaldehyde and
0.1 M sodium cacodylate buffer pH 7.2) for 72 h at
room temperature and dehydrated in ethanol. They
were then immediately desiccated in liquid CO2with a
critical point drier (Hitachi HCP-2), and coated with
palladium-gold in a sputter coater (Eiko IB-2). Finally,
the samples were studied in a scanning electron
microscope (Leo-420).
Chemical microanalysis by Energy Dispersive X-ray
(EDX). In order to analyse their chemical composi-
tion, rays were excised from dorsal fins of healthy
fish, and mechanically scraped to remove the exter-
nal soft tegument. The rays were dried in an oven,
mounted in stubs, carbon coated in a Polaron
CC7650, and observed in a JEOL-6100 scanning
electron microscope with EDX microanalysis. Two
types of measurements were taken: (1) microanalysis
at a point, (2) microanalysis of the average values
in an area.
RESULTS
Experimental infection
Experimental infection was successful. Although
external lesions were not conspicuous in the studied
post-infection times, SEM observation showed the
presence of bacteria in fish tissues, as described below.
SEM observation
In samples where fin rays were not cut, they were cov-
ered by a mucous layer embedding the bacteria (Fig. 1).
80

Martínez et al.: SEM of experimental infection of Flavobacterium psychrophilum
Flavobacterium psychrophilum were rod shape, up to
2µm in length and 0.4 µm in width.
In samples where fin rays were cut immediately
before chemical fixation, the tegument appeared to be
retracted in the basal zone, and the fin axis was naked
and lacking a mucous layer (Fig. 2). In these cases, a
large number of bacteria on the hard material of the fin
axis could be observed, while none were found on the
retracted epidermis. High magnifications of SEM
images showed some of the bacteria lying on the mate-
rial axis, and apparently penetrating directly into the
substrate (Fig. 3).
In samples of 24 or 48 h PIT, the ray axis was fully
eroded by grooves and tubular boreholes whose
dimensions corresponded to that of Flavobacterium
psychrophilum. Bacterial rods could be seen in these
perforations (Fig. 4).
Chemical microanalysis by EDX
Rays from dorsal fin were segmented and occasion-
ally bifurcated (Fig. 5). EDX microanalysis of the ray
axes showed that they were mineralised. Both in punc-
81
Fig. 1. Salmo salar. Uncut ray of the dorsal fin coated by a dense
mucous layer. Post infection time (PIT): 0 h. Scale bar = 10 µm.
Insert: Flavobacterium psychrophilum rods (arrow) embedded in
mucus. PIT: 0 h. Scale bar = 1 µm
Fig. 2. Salmo salar. Ray cut immediately before fixation. The tegu-
ment appears withdrawn to the base of the ray. Numerous
Flavobacterium psychrophilum bacteria can be seen on the ray axis
(see Fig. 3), but not on the epidermis. PIT: 48 h. Scale bar = 10 µm
Fig. 4. Surface of a Salmo salar ray axis eroded by tubular grooves
and boreholes of circular section. PIT: 24 h. Scale bar = 3 µm. Insert:
Note the presence of bacterial rods in the grooves (arrow). PIT: 24 h.
Scale bar = 1 µm
Fig. 3. Flavobacterium psychrophilum bacteria spreading on the
surface of the Salmo salar ray axis of the dorsal fin. PIT: 48 h. Scale
bar = 10 µm. Insert: Rods of F. psychrophilum on the naked axis of
the ray. Some of them seem to be penetrating into the substrate
(arrows). PIT: 24 h. Scale bar = 1 µm
1
3
24

Dis Aquat Org 59: 79–84, 2004
tual and area measurements (Fig. 5), the spectra
showed the presence of significant amounts of Ca
(18.18 and 16.54 in atomic percent, respectively) and
P, besides C and O (corresponding to organic sub-
stances, in general) and traces of Mg and S (Fig. 6).
DISCUSSION
SEM images showed that bacteria spread on the
naked axis of the fin but were absent on the adja-
cent epidermis. This suggests that Flavobacterium
psychrophilum shows a preference in choice of sub-
strate, which had not been reported in previous
studies. Kondo et al. (2002) noted that F. psychrophilum
adhered to the comb-like teeth of infected ayu Pleco-
glossus altivelis and also to the lower jaw and caudal
peduncle, where the epidermis tissue collapsed, but
from their SEM images direct contact of bacteria with
the hard tissues was not observed. Nematollahi et al.
(2003b) showed adherence properties of F. psychro-
philum to the gill arch of rainbow trout Oncorhynchus
mykiss but no electron microscopic images were
presented.
Flavobacterium psychrophilum usually only invades
previously damaged tissue, typically an area of erosion
on the edge of the fins and tail. Infection then pro-
gresses to involve the complete fin or tail and caudal
peduncle (Southgate 1993, Shotts & Starliper 1999).
This interpretation seems to be in agreement with the
SEM images, which suggest that the integrity of the
ray tip plays an important role in protection against F.
psychrophilum invasion. As long as the epidermis and
a thick mucous layer covered the tips, the bacteria had
difficulty accessing the ray axis. Any factor (mechani-
cal or physiological) altering the integrity of that cover-
ing will allow the bacteria to reach the ray axis, which
is the preferred substrate. This would also explain how
the stress imposed by management methods undoubt-
edly predisposes hatchery-raised fish to such infec-
tions (Anderson & Conroy 1969). Mechanical injury of
the fins has been indicated as a plausible entrance for
F. psychrophilum into the fish, and it has been experi-
mentally demonstrated that skin and skin mucus abra-
sion dramatically enhance the invasion of bacteria
(Madetoja et al. 2000). In the later stages of disease,
bacteria also destroy the skin and other tissues, affect-
ing internal organs (Wood & Yasutake 1956, Noga
1996, Shotts & Starliper 1999), but SEM images seem to
indicate that in the initial phase of the process, the first
substrate affected is the ray axis. In advanced cases, or
in recovered fish, bone diseases often develop: scolio-
sis, cranial and vertebral lesions including subacute to
chronic periostitis and ostitis, cephalic osteochondritis
and necrotic scleritis, or inflammation and cartilage
necrosis along the vertebral column (Dalsgaard 1993,
Bruno & Poppe 1996, Ostland et al. 1997, Shotts & Star-
liper 1999, Bader & Starliper 2002). Affected fish may
82
Fig. 5. Bifurcated ray of the Salmo salar dorsal fin. Segments can
be seen in the distal tip (left). The ray was excised and scraped to
remove the external soft tissues. Scale bar = 1 mm. Insert: Point (1)
and Area (2) of the ray axis where EDX (Energy Dispersive X-ray)
microanalyses were carried out. Scale bar = 100 µm
Fig. 6. Results of the EDX (Energy
Dispersive X-ray) microanalysis in
Point (1) and Area (2) (see Fig. 5)
tested on the ray axis of the Salmo
salar fin. Both spectra show a signifi-
cant amount of Ca and P, besides C
and O and traces of Mg and S. cts: cm
5
1
2
Spectrum O Mg P S Ca Total
167.62 0.73 12.74 0.74 18.18 100
271.13 11.90 0.43 16.54 100
All results in Atomic Percent

Martínez et al.: SEM of experimental infection of Flavobacterium psychrophilum
also develop neurological diseases, ataxia and abnor-
mal swimming behaviour, presumably from the locali-
sation of bacteria in the cranium (Noga 1996, Bader &
Starliper 2002).
SEM images showed bacteria covered with mucus.
Bacteria may be destroyed by antimicrobial products
(lysozymes, complement, agglutinins) present in the
mucus (Ellis 1981, Alexander 1985, Yano 1996, Lebe-
deva 1999), but Denkin & Nelson (1999) also demon-
strated that growth or incubation of Vibrio anguillarum
in salmon intestinal mucus rapidly and specifically
induced protease activity. Extracellular proteases have
been shown to be virulence factors for a variety of bac-
teria, including Flavobacterium psychrophilum (Pacha
1968, Otis 1984, Dalsgaard 1993, Bertolini et al. 1994,
Ostland et al. 2000), and they participate in tissue dam-
age to the host. The formation of grooves and tubular
boreholes observed by SEM in the ray axis may be
explained by the effect of products secreted by the
bacteria. Secades et al. (2001) purified and charac-
terised an extracellular protease from F. psychroph-
ilum, designated Fpp1, which was found to be a
55 kDa psychrophilic protein and a potent enzyme
with broad specificity for degrading protein con-
stituents of connective and muscular tissues. This sug-
gests that it participates in pathogenesis by contribut-
ing to colonisation and/or invasion of the fish tissues.
Moreover, the authors showed that the presence of
calcium was necessary for Fpp1 production. The EDX
microanalysis showed the presence of high amounts
of Ca and P in the ray axis, confirming the latter’s
mineralised nature. This suggests that F. psychro-
philum could digest the substrate with the metallo-
protease, and simultaneously it could obtain from the
substrate the necessary Ca for the activation of the
enzyme. This could be the reason for the preference
that bacteria show for the fin rays in early phases of
the infection.
The effect of proteases released by the bacteria is
auniform digestion of the substrate. Nevertheless,
the SEM images showed more individualised effects,
appearing as tubular perforations of dimensions simi-
lar to rods of Flavobacterium psychrophilum, which
seems to indicate that the enzyme has a short opera-
tional range. The perforations appear as circular ori-
fices, indicating that movement of the bacteria is per-
pendicular to the surface. The results suggest the
existence of a mechanical perforation working in con-
junction with the substrate degradation produced by
the chemical processes. The gliding motility, charac-
teristic of this group of bacteria and defined as the
movement of a non-flagellated cell in the direction of
its long axis on a surface (Henrichsen 1972), would
play a role in producing the observed pattern of perfo-
ration. Several models for gliding have been proposed
for different organisms, including, among others,
rotary motors (Pate & Chang 1979), directional extru-
sion of slime (Hoiczyk & Baumeister 1998) and con-
trolled release of surfactants from poles of cells (Keller
et al. 1983). In Cytophaga sp., during gliding in either
the forward or reverse direction, cells were observed
entering into abrupt clockwise and counterclockwise
rotations around either cell pole (Lapidus & Berg
1982), and the entire length of the cell body was rarely
seen in contact with the substratum (Godwin et al.
1989). Whether or not the characteristics of bacterial
movement are implicated in the type of perforation
observed in the fin rays cannot be determined from
SEM images alone.
Acknowledgements. We thank R. Silva and C. Lizama of the
Facultad de Medicina (Universidad Austral de Chile) for tech-
nical assistance in the processing of SEM samples. This work
was made possible by a grant of AECI (Agencia Española de
Cooperación Internacional) to J.L.M.
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84
Editorial responsibility: David Bruno,
Aberdeen, UK
Submitted: October 29, 2003; Accepted: February 4, 2004
Proofs received from author(s): April 1, 2004