Journal of Fish Diseases 2006, 29, 441–444
Short communication
Colonization of rainbow trout, Oncorhynchus mykiss
(Walbaum), eggs by Flavobacterium psychrophilum,
the causative agent of rainbow trout fry syndrome
I N Vatsos1, K D Thompson2 and A Adams2
1 Institute of Aquaculture, Hellenic Centre for Marine Research, Athens, Greece
2 Aquatic Vaccine Unit, Institute of Aquaculture, University of Stirling, Stirling, UK
Keywords: colonization, eggs, Flavobacterium psy-
chrophilum, immunofluorescent antibody tech-
nique, rainbow trout, rainbow trout fry syndrome.
The mucous layer of eggs appears to be a good
substance for adhesion and colonization by many
different bacterial species (Nelson & Ghiorse
1999). Fish pathogens can be transmitted via the
eggs horizontally through water, or vertically from
the broodstock. In the first case, most bacterial
species appear to colonize the surface of the eggs,
but some can also penetrate the eggshell as in the
case of Tenacibaculum ovolyticus (Bergh, Skiftesvik,
Hjeltnes & Roedseth 1994), causing high egg
mortalities.
In the study presented here, rainbow trout fry
syndrome (RTFS)-free rainbow trout eggs were
experimentally infected with Flavobacterium psy-
chrophilum during fertilization. The eggs were
colonized by the pathogen after a bath challenge
and the effect of the pathogen on the eggs was
assessed using an immunofluorescent antibody
technique (IFAT) and transmission electron micr-
oscopy (TEM).
Flavobacterium psychrophilum, isolate B97026,
isolated from a farm in the UK, where RTFS was
endemic, was used to artificially infect rainbow
trout, Oncorhynchus mykiss (Walbaum), eggs.
Approximately 900 unfertilized rainbow trout eggs
Correspondence I Vatsos, Hellenic Centre for Marine Research,
Ag Kosmas, Elliniko, Athens TK 16777, Greece
(e-mail: ivatsos@ath.hcmr.gr)

2006
Blackwell Publishing Ltd 441
and milt were collected from Houghton Springs
Hatchery, Dorset, UK, a site with no previous
reports of RTFS. The eggs were all collected from
one female, put into a plastic bag and sent by mail
to the Institute of Aquaculture, University of
Stirling, UK. Upon arrival and prior to fertilization,
a sample of 20 eggs (selected randomly) was tested
for the presence of F. psychrophilum by nested
polymerase chain reaction (PCR) according to the
method described below. A sample of five eggs was
tested by culture in modified Cytophaga broth
(Bernardet & Kerouault 1989). The milt was not
tested by nested PCR or by culture for the presence
of F. psychrophilum.
The eggs were split into two groups of approxi-
mately 420 each. One group was fertilized, while
the second group was not. Each group was further
divided into two subgroups; one group of approxi-
mately 120 eggs was used as a negative control and
the other group of approximately 300 eggs was
infected with F. psychrophilum (positive group).
This was carried out by mixing together the bacteria
and the eggs.
Fertilization was performed by carefully mixing
the eggs with a few millilitres of milt. The infection
of the eggs was performed as follows: the eggs were
left with the milt for 20 min. Eggs from the positive
groups were carefully mixed with 10 mL bacterial
suspension (3 · 108 mL)1 bacteria in broth). After
30 min, the eggs were placed into small metallic
trays, in a single layer, and the trays were then put
into 20-L plastic tanks until hatching. The tanks
were supplied constantly with fresh water. The
 Journal of Fish Diseases 2006, 29, 441–444 I N Vatsos et al. Colonization of rainbow trout eggs by F. psychrophilum
group of eggs not infected with F. psychrophilum
(negative control) was transferred to the tanks after
50 min. The water temperature during incubation
of the eggs was 11 C at the beginning of the
experiment and was gradually decreased to 7.8 C
by the end of the experiment. Samples of eggs were
collected on days 1, 5, 10 or 30 post-fertilization
and examined by IFAT and TEM (for both
methods, 10 eggs were collected from the infected
groups and five eggs from the negative controls).
On the same days, samples of eggs were also
examined by culture (five eggs per group) and
nested PCR (20 eggs per group) to confirm the
presence (infected groups) or absence (uninfected
groups) of the pathogen throughout the experi-
mental period. After rinsing the eggs in sterile
0.02 m phosphate-buffered saline (PBS, pH 7.2)
and squashing them with a needle, they were placed
in Cytophaga broth for 4 days at 15 C. After this
time, four 100-lL aliquots were sampled from each
tube and spread onto Cytophaga agar plates and the
plates incubated until yellowish colonies were
visible. Colonies were collected and re-suspended
in 500 lL PBS. Drops of each suspension were
placed on slides, air-dried and stained with Gram’s
stain. The colonies were also screened by nested
PCR, after re-suspending them in 1 mL PBS.
Nested PCR analysis (Vatsos, Thompson &
Adams 2003), using the primers developed by
Toyama, Kita-Tsukamoto & Wakabayashi (1994),
was carried out on the aqueous part of the
homogenized egg samples, following centrifugation
at 3000 g for 15 min.
For the IFAT, the eggs were first fixed for 24 h
using 10 mL glacial acetic acid, 5 mL formaldehyde
(30% solution) and 0.6 m calcium chloride, and
were then dehydrated at 20 C in an alcohol series:
1 day in each of 70%, 80%, 90% and 95% ethanol.
A commercially available kit (Technovit 7100,
TAAB Kit; Energy Beam Sciences, East Granby,
CT, USA) was used for the infiltration and
embedding of the samples. After dehydration, the
eggs were infiltrated with the ÔliquidÕ base of the kit,
using 50:50 base: 95% ethanol at 20 C for 3 days,
75:25 base: 95% ethanol for 5 days and 100% base
together with hardener I (infiltration solution, 1 g
hardener I per 100 mL base) for 7 days at 4 C.
The eggs were finally embedded in resin (1 mL of
hardener II mixed in 15 mL of infiltration solution)
using Teflon-coated moulds. Once the resin had
hardened, the egg samples were mounted on
wooden stubs. Three sections, 2-lm thick, were

2006
Blackwell Publishing Ltd 442
cut from each sampled egg using a Reichert
Ultracut E (Leica, Vienna, Austria) microtome
fitted with a glass blade and sections were then
transferred into a 50 C water bath to stretch them.
Sections were finally mounted onto clean slides and
left to air dry. The IFAT was performed according
to the method described by Vatsos, Thompson &
Adams (2002) using anti-F. psychrophilum (isolate
B97026) rabbit serum diluted in PBS. The sections
were observed under immersion oil using an
Olympus fluorescence microscope (Olympus
Optical Co Ltd., Tokyo, Japan).
The egg samples collected for TEM were processed
according to the method described by Vatsos et al.
(2002). At least three sections were cut per egg.
Many microorganisms colonizing the surface of
eggs have adverse effects on their development, as
observed by Bergh, Hansen & Taxt (1992), who
experimentally infected halibut, Hippoglossus hippo-
glossus (L.), eggs with bacteria of the genera
Flexibacter and Vibrio. Factors such as water
temperature, egg density and poor water flow also
seem to promote bacterial overgrowth on the eggs
(Barker, Smith & Bromage 1989). The dead or
damaged eggs can leak nutrients aiding bacterial
multiplication (Barker et al. 1989; Ogbondeminu
1994).
In the present study, a large number of microbes
on the surface of eggs of all groups were observed by
TEM (Fig. 1b). The surface of the unfertilized eggs
appeared more heavily colonized by bacteria than
fertilized eggs and in a few instances the mucous layer
of infected eggs appeared to be partially destroyed,
especially in eggs collected just before hatching.
Although it was not possible to identify F. psychro-
philum in the sections using TEM, it is believed that
this destruction of the mucous layer was due to
enzymatic activity of the pathogen, as such areas were
not found in uninfected eggs. Uddin & Wakabayashi
(1997) found F. psychrophilum to exhibit high levels
of proteolytic activity at low temperatures (around
13.3
1.9 C), especially during the late log or
stationary phase of its growth cycle.
Throughout the course of the experiment, mixed
colonies were seen on agar plates in samples
collected from all groups of eggs. In samples of
infected eggs taken from both fertilized and unfer-
tilized groups, a few yellowish colonies could also be
seen on the agar plates. Bacteria collected from
these yellowish colonies were identified as F. psy-
chrophilum, according to their morphological char-
acteristics and after nested PCR analysis.
 Journal of Fish Diseases 2006, 29, 441–444
(a)
Figure 1 (a) Identification of Flavobacterium psychrophilum (white arrow) on the surface of an infected fertilized rainbow trout egg using
immunofluorescent antibody technique, 30 days post-fertilization (bar ¼ 10 lm). (b) Transmission electron microscopy. Mixed
microflora on the surface of an infected fertilized rainbow trout egg; destruction of mucous layer (*) by proteolytic bacteria, 30 days
post-infection (bar ¼ 10 lm).
Only in infected groups of eggs could patho-
gens be seen on the egg surface using IFAT
(Fig. 1a). It should be noted that the numbers of
F. psychrophilum on the surface of the eggs was
low (one to three bacterial cells per observation
area). Overgrowth of the pathogen on the surface
of the eggs may have been prevented as no adverse
environmental conditions (such as reduced water
flow) co-existed, a parameter that other authors
have suggested as critical for the effect bacterial
pathogens may have on the surface of the eggs
(Barker et al. 1989; Barnes, Gabel & Cordes
2000). Although the antiserum used is known to
cross-react with a few other bacterial species
(Faruk, Campbell, Thompson, Rangdale &
Richards 2002), positive results were obtained only
in the infected groups. In a similar experiment,
Kumagai, Yamaoka, Takahashi, Fukuda & Waka-
bayashi (2000) examined frozen egg sections by
IFAT taken from fertilized coho salmon, Oncor-
hynchus kisutch (Walbaum), eggs immersed into
broth culture of F. psychrophilum. They found
that some bacteria had managed to penetrate the
eggs in sections observed 57 days post-infection.
However, it may have been that the bacteria
spread to the inside of the egg due to the cutting
of the frozen sections, thus producing a false
picture of their location within the egg. On the
other hand, Kumagai, Nakayasu & Oseko (2004)
concluded that F. psychrophilum did not penetrate
the surface of ayu, Plecoglossus altivelis (Temminck
& Schlegel), eggs, which were colonized by the
bacterium.

2006
Blackwell Publishing Ltd 443
I N Vatsos et al. Colonization of rainbow trout eggs by F. psychrophilum
(b)
Previous studies suggested that broodstock is an
important source of contamination of eggs with
F. psychrophilum (Cipriano 2005). Nevertheless,
waterborne colonization of the surface of the eggs
is important as it can lead to RTFS (Rangdale,
Richards & Alderman 1997) and act as an
additional factor for the spread of the disease
between farms (Kumagai & Takahashi 1997).
The results of this study suggest that, after a
waterborne colonization of the surface of the eggs,
F. psychrophilum remains on the egg surface and
does not appear to penetrate, although areas of
destruction of the mucous layer occur. Different
isolates of F. psychrophilum and different physio-
logical states of the bacterium, such as late log
phase or after starvation, should also be tested,
since previous studies showed that under condi-
tions of starvation many functions of F. psychro-
philum are altered and the bacterium adheres to
the eggs in higher numbers, compared with
F. psychrophilum collected from fresh cultures
(Vatsos, Thompson & Adams 2001; Vatsos et al.
2003).
Acknowledgements
The authors would like to thank Dr Ali Reza Faruk,
Department of Aquaculture, Bangladesh Agricul-
ture University, Mymensingh, Bangladesh for pro-
viding the antibodies used in this study and
Mr Linton Brown, Institute of Aquaculture,
University of Stirling, for his guidance on the
electron microscopy techniques.
 Journal of Fish Diseases 2006, 29, 441–444 I N Vatsos et al. Colonization of rainbow trout eggs by F. psychrophilum
References
Barker G.A., Smith N. & Bromage N.R. (1989) The bacterial
flora of rainbow trout, Salmo gairdneri Richardson, and brown
trout, Salmo trutta L., eggs and its relationship to develop-
mental success. Journal of Fish Diseases 12, 281–293.
Barnes M.E., Gabel A.C. & Cordes R.J. (2000) Bacterial
populations during rainbow trout egg culture in vertical-flow
tray incubators. North American Journal of Aquaculture 62,
48–53.
Bergh Ø., Hansen G.H. & Taxt R.E. (1992) Experimental
infection of eggs and yolk sac larvae of halibut, Hippoglossus
hippoglossus (L). Journal of Fish Diseases 15, 379–391.
Bergh Ø., Skiftesvik A., Hjeltnes B. & Roedseth O. (1994)
Pathogen–host relations between bacteria and marine fish eggs
and larvae. Third International Marine Biotechnology
Conference, Tromso, Norway, 7–12 August 1994, p. 92.
Bernardet J.F. & Kerouault B. (1989) Phenotypic and genomic
studies of ÔCytophaga psychrophilaÕ isolated from diseased
rainbow trout (Oncorhynchus mykiss) in France. Applied and
Environmental Microbiology 55, 1796–1800.
Cipriano R.C. (2005) Intraovum infection caused by Flavobac-
terium psychrophilum among eggs from captive Atlantic salmon
broodfish. Journal of Aquatic Animal Health 17, 275–283.
Faruk M.A.R., Campbell R.E., Thompson K.D., Rangdale R.E.
& Richards R.H. (2002) Characterisation of Flavobacterium
psychrophilum, the causative agent of rainbow trout fry syn-
drome (RTFS), using rabbit serum. Bulletin of the European
Association of Fish Pathologists 22, 354–365.
Kumagai A. & Takahashi K. (1997) Imported eggs responsible
for the outbreaks of cold-water disease among cultured coho
salmon in Japan. Fish Pathology 32, 231–232.
Kumagai A., Yamaoka S., Takahashi K., Fukuda H. &
Wakabayashi H. (2000) Waterborne transmission of
Flavobacterium psychrophilum in coho salmon eggs. Fish
Pathology 35, 25–28.

2006
Blackwell Publishing Ltd 444
Kumagai A., Nakayasu C. & Oseko N. (2004) No evidence for
the presence of Flavobacterium psychrophilum within ayu eggs.
Fish Pathology 39, 183–188.
Nelson E.J. & Ghiorse W.C. (1999) Isolation and identification
of Pseudoalteromonas piscicida strain Cura-d associated with
diseased damselfish (Pomacentridae) eggs. Journal of Fish
Diseases 22, 253–260.
Ogbondeminu F.S. (1994) Commensal bacterial microflora
associated with incubating eggs of Clarias anguillaris in a tropical
hatchery. Journal of Aquaculture in the Tropics 9, 151–156.
Rangdale R., Richards R. & Alderman D. (1997) Colonisation
of eyed rainbow trout ova with Flavobacterium psychrophilum
leads to rainbow trout fry syndrome in fry. Bulletin of the
European Association of Fish Pathologists 17, 108–111.
Toyama T., Kita-Tsukamoto K. & Wakabayashi H. (1994)
Identification of Cytophaga psychrophila by PCR targeted 16S
ribosomal RNA. Fish Pathology 29, 271–275.
Uddin M. & Wakabayashi H. (1997) Effects of temperature on
growth and protease production of Cytophaga psychrophila.
Fish Pathology 32, 225–226.
Vatsos I.N., Thompson K.D. & Adams A. (2001) Adhesion of
the fish pathogen Flavobacterium psychrophilum to unfertilized
eggs of rainbow trout (Oncorhynchus mykiss) and
n-hexadecane. Letters in Applied Microbiology 33, 178–182.
Vatsos I.N., Thompson K.D. & Adams A. (2002) Development
of in situ hybridization and immunofluorescent antibody
technique (IFAT) to detect Flavobacterium psychrophilum, in
water samples. Aquaculture Research 33, 1087–1090.
Vatsos I.N., Thompson K.D. & Adams A. (2003) Starvation
of Flavobacterium psychrophilum in broth, stream water and
distilled water. Diseases of Aquatic Organisms 56, 115–126.
Received: 21 November 2005
Revision received: 27 March 2006
Accepted: 1 May 2006