Directory UMM :Data Elmu:jurnal:A:Aquaculture:Vol183.Issue1-2.Mar2000:
Aquaculture 183 Ž2000. 31–44
www.elsevier.nlrlocateraqua-online
Vaccination of Atlantic halibut Hippoglossus
hippoglossus L., and spotted wolffish Anarhichas
minor L., against atypical Aeromonas salmonicida
Marina Ingilæ, Jan Arne Arnesen, Vera Lund ) , Guri Eggset
1
Fiskeriforskning, Norwegian Institute of Fisheries and Aquaculture, N-9291 Tromsø, Norway
Abstract
Atypical furunculosis caused by atypical Aeromonas salmonicida is an increasing problem in
commercial halibut farming, and a potential problem in farming of spotted wolffish in Norway.
Halibut, Hippoglossus hippoglossus L., and spotted wolffish, Anarhichas minor L., vaccinated
with oil-emulsified vaccines against atypical furunculosis, demonstrated a relative percent survival
ŽRPS. of approximately 90 when challenged with homologous isolates. Bath and aqueous injection
vaccines failed to protect against disease when challenged 10 weeks post-vaccination. High
antibody titres were produced in both species after vaccination with oil-emulsified vaccines, and
the major antibody response were against A-layer, LPS and some minor outer membrane ŽOM.
proteins. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Atypical Aeromonas salmonicida; Furunculosis; Vaccination; Halibut; Hippoglossus hippoglossus
L.; Spotted wolffish; Anarhichas minor L.
1. Introduction
The species Aeromonas salmonicida is, according to Bergey’s Manual of Determinative Bacteriology Ž1994., divided into three subspecies: subsp. salmonicida ŽGriffin et
al., 1953., subsp. achromogenes ŽSmith, 1963., and subsp. masoucida ŽKimura, 1969..
Other subspecies have been suggested as subsp. smithia ŽAustin et al., 1989. in addition
)
C orresponding author. T el.: q 47-77-62-90-00; fax: q 47-77-69-91-00;
vera.lund@fiskforsk.norut.no
1
Present address: Tromsø Science Park, Forskningsparken, N-9291, Tromsø, Norway.
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 2 7 9 - 3
e-m ail:
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M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
to an increasing number of reported strains that have not been assigned to any of these
subspecies. A. salmonicida subsp. salmonicida comprises a homogenous group of
strains and are referred to as typical A. salmonicida causing classical furunculosis,
while all other A. salmonicida strains are referred to as atypical and comprise a
heterogenous group causing diseases referred to as ulcer disease or atypical furunculosis
Žreviewed by Wiklund and Dalsgaard, 1998..
The number of published reports of disease outbreaks associated with atypical strains
of A. salmonicida has increased significantly during the last decade, and these isolates
have been reported from an increasing number of fish species and geographical areas
Žreviewed by Wiklund and Dalsgaard, 1998.. The virulence of atypical A. salmonicida
varies according to the host species, and variations in susceptibility to different strains of
atypical A. salmonicida are observed in both salmonids and non-salmonids. Some of the
atypical strains isolated from wild fish adversely affect salmonids and therefore may
represent a significant risk to farmed fish. At present, several non-salmonid fish species
have been introduced into farming, while other possible species are being tested for that
purpose. The susceptibility of these fish species to atypical A. salmonicida has to be
examined. Atypical furunculosis is an increasing problem in commercial halibut farming, and a potential problem in farming of spotted wolffish in Norway. Wild wolffish
caught and used as breeding stocks may be carriers of the bacterium, and outbreaks
frequently occur when the fish are stressed or when water temperature increases above
88C–108C ŽHellberg et al., 1996.. Commercial furunculosis vaccines based on A.
salmonicida subsp. salmonicida and produced for the prevention of furunculosis in
salmonids, did not give the same degree of protection against disease caused by atypical
bacterial strains in salmonids ŽGudmundsdottir and Gudmundsdottir, 1997., and probably neither against atypical furunculosis in non-salmonids. The aim of the present study
was to examine whether vaccines based on bacterial strains isolated from diseased
halibut and wolffish, may yield protective effect against atypical furunculosis in these
fish species.
2. Materials and methods
2.1. Fish
Halibut, Hippoglossus hippoglossus L. Ž; 15 g. and spotted wolffish, Anarhichas
minor L. Ž; 39 g in experiment 2 and ; 28 g in experiment 3, see Table 1. produced
from wild caught breeding stocks were used in this study. The halibut were obtained
from Lofilab, Leknes, Norway, while the wolffish were bred at the Aquaculture
Research Station in Tromsø, Norway, where the vaccination and experimental challenges were performed. The halibut were kept at 128C in a 500 l tank, and the wolffish
in raceways measuring 40 = 17 = 210 cm and 20 = 20 = 100 cm in experiments 2 and
3, respectively, both at 108C during the experiments. Prior to all handling such as
injection of vaccines or challenge bacteria or individual weighing, the fish were
anaesthetized with benzocain Ž50 mgrl water.. The different groups were spotmarked
with Alcian blue ŽPanjet..
Vaccine type
Administration
Dose
Aqueous vaccinea
Aqueous vaccinea
FIA-emulsifieda
Alpharma-emulsifieda
Unvaccinated control
Challenge method
Challenge dose
Immersion
i.p. injection
i.p. injection
i.p. injection
5=10 8 bacteriarml
2.5=10 9 bacteriarfish
2.5=10 9 bacteriarfish
2.5=10 9 bacteriarfish
a
Experiment 1 Žhalibut.
Experiment 2 Žwolffish.
Experiment 3 Žwolffish.
Fish per group
Fish per group
Fish per group
50 b
50
50
50 c
50
50
50
Bath 60 min
10 5 LFI 4050rml
50
i.p. injection
10 7 LFI 4048rfish
The halibut and the spotted wolffish vaccines contained the A. salmonicida isolates LFI 4050 and LFI 4048, respectively.
Immersion for 15 min.
c
Immersion for 45 min.
b
50
50
i.p. injection
10 4 LFI 4048rfish
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
Table 1
Experimental design
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M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
2.2. Bacteria
Two atypical A. salmonicida strains, LFI 4050 isolated from diseased halibut, and
LFI 4048 from diseased spotted wolffish, were used for vaccination and challenge of
halibut and spotted wolffish, respectively. They were shown to be different by biochemical tests ŽAPI 20E and API 50 CH., and by production of extracellular proteases
demonstrated in substrate gels. In addition, the strain LFI 4061 isolated from another
outbreak of atypical furunculosis in the breeding stock of spotted wolffish was used for
challenge. A typical A. salmonicida subsp. salmonicida LFI 4045 was used as a
reference strain in Western blots.
The bacteria used in the vaccines and for challenge were grown in Brain heart
infusion broth ŽBHI, Difco. containing 2% NaCl at 128C for 24 h with shaking. The
cells used in vaccines were inactivated by addition of 0.5% vol.rvol. formaldehyde
solution Ž37%. for 48 h, washed in PBS Ž20 mM phosphate, 150 mM NaCl, pH 7.4. and
resuspended in PBS before used to prepare the different vaccines ŽTable 1.. BHI agar
ŽOxoid., supplemented with 0.005% Coomassie brilliant blue, and Oxoid blood agar
base no. 2 supplemented with 2% human red blood cells and 1.5% NaCl, were used for
reisolation of bacteria from dead fish.
2.3. Vaccines and Õaccination
Three vaccination and challenge experiments were performed, one on halibut and two
on spotted wolffish. Each experiment was run in separate tanks or raceways. The
experimental design is summarised in Table 1. Four different vaccines were tested for
protection against atypical furunculosis; one immersion and three injection vaccines. The
bacterial stock suspension described above were diluted in order to obtain appropriate
cell concentrations in the different vaccines. The immersion vaccines were diluted in sea
water, the aqueous injection vaccines in PBS, and in the oil-emulsified vaccines the
bacterial suspension was diluted in PBS before emulsification in the oil adjuvant in
proportion 1:1 ŽTable 1.. The adjuvant used was either Freunds incomplete adjuvant
ŽFIA-emulsified. or the vegetable–animal oil adjuvant ŽAlpharma-emulsified. used in
the commercial furunculosis vaccines for Atlantic salmon ŽApoject-Fural vaccines,
Alpharma, Norway.. In 1998 the Apoject vaccines were replaced with the Alphaject
series which instead contains a mineral oil adjuvant. The vaccines used for the halibut
and spotted wolffish contained the A. salmonicida strains LFI 4050 and LFI 4048,
respectively. Fifty fish of each group were vaccinated either by immersion or by
intraperitoneal Ži.p.. injection of 0.1 ml of vaccines, and one group of 50 unvaccinated
fish was left as control in each experiment. In experiment 3 ŽTable 1., each fish was
weighed at the time of vaccination and challenge.
2.4. Prechallenge and challenge of fish
Challenge was performed 10 weeks post-vaccination, unless otherwise stated, with
the same A. salmonicida strains as used in the vaccines Žhomologous strains.. The
challenge method used on spotted wolffish was determined in a prechallenge experi-
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
35
ment, where groups of 10 fish were challenged with different bacterial doses by bath Ž60
min. or i.p. injection of 0.1 ml bacterial suspension. The bath and i.p. infected groups
were kept in the same raceway as an uninfected group to be infected through the water
by cohabitation.
Bath challenge, using the same bacterial concentration as had successfully been used
in previous experiments, was performed on halibut. The water level in the tank was
lowered to 50 l and the flow stopped before addition of the bacterial suspension. The
water was oxygenated during challenge.
Mortality in each group was recorded daily. The cause of death was verified by
reisolation of bacteria from kidney samples of dead fish. Cumulative mortality was
registered and relative percent survival ŽRPS. on the last day of the experiment was
calculated according to the formula: RPS s w1 y % mortality in vaccinated
groupr%mortality in control groupx = 100%..
The fish in experiment 3 surviving challenge with homologous bacterial strain,
together with a group of 50 unvaccinated, previously unchallenged fish of approximately
the same size and from the same batch, were challenged 15 weeks after the first
challenge with a heterologous A. salmonicida strain LFI 4061.
2.5. Humoral immune responses
Ten additional fish of each group in experiments 1 and 2 were vaccinated to study
specific antibody responses. The fish were sampled at the time of challenge and blood
was drained. Specific antibody responses were quantified in the sera collected, using
homologous formalin killed whole A. salmonicida cells in an enzyme-linked immunosorbent assay ŽELISA., as described by Lund et al. Ž1991.. Bound antibodies were
detected with alkaline phosphatase conjugated rabbit anti-wolffish and anti-halibut IgM
produced in the author’s laboratory.
The antibody responses were evaluated qualitatively by Western blotting, using outer
membrane ŽOM. preparations ŽFilip et al., 1973. of the two atypical A. salmonicida
strains LFI 4050 and 4048, and one typical A. salmonicida subsp. salmonicida LFI
4045 strain. The OM preparations were boiled for 5 min in 5% sodium dodecyl sulphate
ŽSDS, Sigma. with 0.2 M dithiothreitol ŽDTT, Sigma. for denaturation and reduction,
prior to separation in a 12% SDS–polyacrylamide gel ŽLaemmli, 1970.. The electrophoretic separation of the OM proteins and the transfer of the proteins ŽWestern blot.
onto a 0.45 mm nitrocellulose membrane ŽTrans-Blot w , Bio-Rad. was performed on a
Mini-PROTEAN II system ŽBio-Rad. according to the Bio-Rad Instruction Manual.
Three parallel gels were run, one was silver stained ŽMorrissey, 1981. and two were
used for Western blotting. Low Molecular Weight markers ŽPharmacia. or Kaleidoscope
markers ŽBio-Rad. were used when gels were silver stained or electroblotted, respectively.
In order to identify OM proteins reacting with the fish antibodies, the membranes
were first blocked with PBS containing 0.05% Tween 20 ŽPBS–Tween. and 5% non-fat
dry milk. Primary antisera, either from halibut or wolffish, were diluted 1:100 in
PBS–Tween with 0.5% non-fat dry milk. Secondary antibodies were the alkaline
phosphatase conjugated rabbit anti-halibut or anti-wolffish IgG mentioned above. The
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M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
membrane was incubated with the different antibodies for 60 min at room temperature
with gentle shaking, and washed 3 = 10 min with PBS–Tween between each incubation
step. Finally, the membranes were stained by adding the substrate nitroblue tetrazolium
chloride and 5-bromo-4-chloro-3-indolylphosphate ŽNBT and BCIP; Gibco BRL. in
substrate buffer Ž0.1 M Tris pH 9.5, 0.1 M NaCl and 50 mM MgCl 2 . as recommended
by the producer. The staining was stopped by washing the membrane in distilled water.
The specificity of the primary antisera had previously been verified by comparing the
reactivity of normal sera and immune sera from both halibut and wolffish to A.
salmonicida OM preparations on Western blot.
3. Results
3.1. Vaccination and challenge
The halibut were challenged by bath, as more than 50% mortality had been obtained
by such challenge Ž10 4 –10 5 cfurml. in previous trials. Prechallenge experiments
performed on wolffish showed that more that 50% of the fish died after injection
challenge with doses corresponding to 10 4 –10 7 cfurml, dependent of fish size, while
bath challenge resulted in mortalities of approximately 20% even at doses of 10 8 cfurml
Ždata not shown.. Challenge by cohabitation was not successful as the fish were not
diseased or died within a time scale of 4 weeks. Thus, injection challenge Ži.p.. was
chosen for spotted wolffish.
In experiments 1 and 2, respectively, groups of halibut and spotted wolffish were
vaccinated with the different vaccine formulations as described in Table 1, and challenged with homologous A. salmonicida strains 10 weeks later. The results are
presented as cumulative mortalities in Fig. 1. The onset of death in the bath challenged
halibut groups started 11–12 days post-challenge for all groups ŽFig. 1a.. The mortality
of the group vaccinated by immersion was similar to that of the unvaccinated group.
Compared to these, the group that obtained aqueous injection vaccine showed a lower
mortality throughout the challenge experiment. However, at the end of the experimental
period Žday 39 post-challenge., all three groups reached similar cumulative mortalities
Ž43%–47%.. On the other hand, the protection obtained in the group vaccinated with
oil-emulsified vaccine ŽFIA-emulsified. was convincing. In this group the mortality
Ž5%. was significantly lower Ž P - 0.05. than those of the other groups at the end of the
experiment, yielding RPS s 89.
The onset of death in the i.p. challenged wolffish groups occurred on day 3
post-challenge ŽFig. 1b.. All vaccinated groups and the unvaccinated control group
reached 100% mortality on day 17 post-challenge, indicating that a too high bacterial
Fig. 1. Experimental challenge of differently vaccinated and unvaccinated groups of halibut and spotted
wolffish 10 weeks post-vaccination ŽTable 1.. ŽA. The atypical A. salmonicida strain LFI 4050, from halibut,
was used in the vaccines and for bath challenge Ž10 5 rml, 60 min.. ŽB. The atypical A. salmonicida strain LFI
4048 from spotted wolffish was used in the vaccines and for i.p. challenge Ž10 7rfish..
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
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38
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
Fig. 2. Experimental challenge of spotted wolffish 10 weeks after i.p. vaccination with A. salmonicida LFI
4048 emulsified in Alpharma’s oil adjuvant. The homologous bacterial strain was used for i.p. challenge Ž10 4
cellsrfish..
dose had been used for infection. However, on day 7, the groups vaccinated with
oil-emulsified vaccine, aqueous injection vaccine, or immersion vaccine showed cumulative mortalities of 38%, 60% and 82%, respectively, indicating the FIA-emulsified
vaccine to be the most promising vaccine candidate.
A second vaccination and challenge experiment Žexperiment 3, Table 1. was performed on wolffish, using Alpharma’s commercial oil adjuvant ŽAlpharma-emulsified.
instead of FIA. Also, a lower bacterial dose was used for this challenge. The results of
the challenge with the homologous A. salmonicida strain are shown in Fig. 2. The
unvaccinated control group started to die 5 days post-challenge, and 90% mortality was
reached on day 12. In the vaccinated group, however, only 14% cumulative mortality
was obtained 26 days post-challenge, corresponding to RPS s 90. Thus, a significant
protection against A. salmonicida was obtained in spotted wolffish when using a
vaccine containing a commercial oil adjuvant. Fifteen weeks later, 43 vaccinated fish
surviving this experiment were subjected to challenge by i.p. infection with a heteroloFig. 3. Specific antibody responses against atypical A. salmonicida in the different vaccinated groups analysed
by ELISA. ŽA. Antibody responses in halibut to strain LFI 4050. ŽB. Antibody responses in spotted wolffish to
strain LFI 4048.
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
39
40
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
gous A. salmonicida strain, isolated from moribund wild caught spotted wolffish. Also,
50 non-vaccinated and hitherto unchallenged fish were challenged as controls. All the
fish in the latter group died, but none in the vaccinated group Ždata not shown.. This
indicates that vaccines based on a single atypical A. salmonicida strain may be
protective against heterologous bacterial strains, at least those pathogenic to the same
fish species.
3.2. Humoral immune responses
The quantitative antibody responses in halibut and spotted wolffish against whole
cells of the A. salmonicida strains used in the vaccines, were determined by ELISA. By
defining the antibody titre as serum dilution yielding an optical density at 405 nm of
; 1, the oil-emulsified and aqueous injection vaccines induced titres of ) 1280 and
; 160, respectively, in both halibut and wolffish, while no specific responses were
observed in sera of immersion vaccinated fish ŽFig. 3..
Qualitative antibody responses of halibut and spotted wolffish to OM antigens of the
two atypical A. salmonicida strains used in the vaccines are shown on Western blots in
Fig. 4. The cross-reactions of these sera with OM antigens of a typical A. salmonicida
strain are also shown. Only small variations between the OM patterns of the two atypical
strains and the typical A. salmonicida strain were observed on the silver stained gel.
Antisera from halibut and spotted wolffish both reacted with the A-layer protein and
HMW LPS in the 50–60 kDa region, and some unidentified proteins with molecular
weights of 35–50 kDa in all three A. salmonicida strains. In addition the halibut
Fig. 4. Qualitative antibody responses in halibut and spotted wolffish. ŽA. Silver stained gel with OM proteins;
lane 1: low molecular weight marker, lane 2: atypical A. salmonicida LFI 4050 isolated from halibut, lane 3:
atypical A. salmonicida LFI 4048 isolated from spotted wolffish, lane 4: typical A. salmonicida subsp.
salmonicida LFI 4045. ŽB. Western blot of parallel gel as in A immunostained with halibut antiserum to A.
salmonicida LFI 4050. ŽC. Western blot of parallel gel as in A immunostained with spotted wolffish antiserum
to A. salmonicida LFI 4048.
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
41
antiserum seems to cross-react with some low molecular weight components Ž; 10 kDa.
in all three isolates.
4. Discussion
Vaccines formulated with oil adjuvants yielded better protection than aqueous
injection or bath vaccines. A protection of approximately 90% RPS could be obtained on
challenge with homologous A. salmonicida strains in both halibut and spotted wolffish
ŽFig. 1a and Fig. 2.. The efficacy of the vaccines correlated with specific antibody
responses, as the highest antibody titres were demonstrated in fish vaccinated with the
oil-emulsified vaccines. Since atypical A. salmonicida strains comprise a heterogeneous
group of bacteria, vaccinated fish should be challenged with several isolates of different
species and geographical locations, in order to determine if more than one isolate is
needed in the vaccines. In this experiment, fish shortage did not allow for challenge with
several bacterial strains. However, in experiment 3, vaccinated wolffish surviving
challenge with homologous bacterial strain, were subjected to a second challenge with a
heterologous strain. The vaccine also protected effectively against this particular strain.
The first challenge might, however, have functioned like a boost and increased the
protection of the vaccinated fish compared to the unvaccinated and previously unchallenged control fish.
The high final mortalities in all wolffish groups in experiment 2 were probably due to
the high challenge dose Ž10 7 cells per fish.. Therefore, a reduced dose Ž10 4 cells per
fish. was used in experiment 3. Even though the mortality of the unvaccinated control
group in the latter experiment also reached nearly 100%, a RPS as high as 89 was
obtained in the vaccinated group.
Vaccination with oil-emulsified vaccines seemed to reduce the growth of both halibut
and spotted wolffish. In experiments 1 and 2, an inhibition of growth was only visually
observed, since the mean weights of each vaccination group were not measured.
Therefore, in experiment 3, the mean weights of the groups were measured both at the
time of vaccination and challenge. The growth of the fish was seriously repressed as the
mean weight of the fish was 28 g when vaccinated, while at challenge 10 weeks later,
the vaccinated and unvaccinated groups were 33 and 76 g, respectively. Also, the
intestines were pale and shrunken compared to the control group, but adherances
between internal organs were rare. However, 5 months post-vaccination, the intestines of
the surviving vaccinated fish were normal and the growth rate Ž%. was the same as for
unvaccinated backup fish of the same batch.
The Alpharma-emulsified vaccine seemed to have a more serious effect on the
growth of the spotted wolffish compared to that observed in Atlantic salmon vaccinated
with Alpharma’s Apoject vaccines containing the same vegetable–animal oil adjuvant
Žinformation from Alpharma.. However, recently a vaccine containing the mineral oil
used in Alpharma’s Alphaject vaccines, was shown to have less effect on the growth of
spotted wolffish Ždata not shown., indicating that the period of growth inhibition can be
reduced.
Atypical and typical A. salmonicida strains share major cell-surface antigens such as
the A-protein layer ŽEvenberg et al., 1985. the LPS component ŽPyle and Cipriano,
42
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
1986., iron-regulated OM proteins ŽHirst and Ellis, 1994. and porins ŽLutwyche et al.,
1995., while the production of exotoxins differs ŽGudmundsdottir, 1996.. In this study,
western blots using OM preparations as antigens showed that both halibut and spotted
wolffish produced antibodies to the A-layer protein, LPS and some unidentified proteins
of homologous isolate. The antisera also cross-reacted with corresponding proteins of
typical A. salmonicida subsp. salmonicida. Little information is available about immunological protection of fish against infection with atypical A. salmonicida. Vaccination against typical furunculosis has been shown to protect against atypical furunculosis
in both salmonids and cyprinid fish ŽJones et al., 1996.. The authors of the present study
have experienced that A-layer protein on bacterial cells is an important protective
antigen in vaccines against typical furunculosis in salmonids, since no protection was
obtained with vaccines containing a bacterial strain lacking the A-layer Žunpublished
data.. Hirst and Ellis Ž1994. found that the iron-regulated membrane proteins of A.
salmonicida represented protective antigens important for successful vaccines against
both typical and atypical strains of A. salmonicida in salmon. Extracellular products
ŽECP. appeared to be important for protection against atypical A. salmonicida in carp
ŽEvenberg et al., 1988. and Atlantic salmon ŽGudmundsdottir and Magnadottir, 1997..
ECP of the subsp. achromogenes elicited better protection than whole bacteria in
Atlantic salmon, and the protection strongly correlated with the detection of antibodies
directed against the 20 kDa extracellular metallocaseinase AsaP1 in fish sera
ŽGudmundsdottir and Magnadottir, 1997..
The only commercial currently available vaccine against atypical furunculosis is an
autogenous emulsion vaccine against A. salmonicida subsp. achromogenes in salmonids
for use in Iceland where the strain is endemic ŽIceland Biojec OO, Alpharma N.W...
Both the autogenous vaccine and a commercial vaccine against classical furunculosis
ŽBiojec1500, Alpharma N.W.. evoked protection against atypical furunculosis in Atlantic salmon, the former yielding an apparently better protection than the latter
ŽGudmundsdottir and Gudmundsdottir, 1997.. On the other hand, no protection against
classical furunculosis was achieved with the autogenous vaccine. This indicate that the
two vaccines contain common antigens capable of yielding some protection against
atypical furunculosis, but insufficient to protect against classical furunculosis. However,
it remains to be tested whether the salmonid vaccine against atypical furunculosis may
protect against atypical A. salmonicida in marine fish species.
Challenge by bath or cohabitation is preferable since they mimic the infection
mechanisms in the environment. In this study, bath challenge was used on halibut, while
injection challenge Ži.p.. was used on spotted wolffish. Bath or cohabitation infection
was not successfully established in prechallenge experiments on the latter species. Both
bath and cohabitation models are commonly used, both in fresh and seawater, when
challenging commercial vaccines against typical furunculosis in Atlantic salmon. However, using A. salmonicida subsp. achromogenes, Gudmundsdottir and Gudmundsdottir
Ž1997. were only able to establish an intra muscular infection in Atlantic salmon in fresh
water. The necessity of using different challenge methods on halibut and wolffish is not
readily explained. It may reflect that farmed halibut is more susceptible to infection by
atypical A. salmonicida than farmed wolffish. Farmed halibut, inhabiting the bottom of
the tank may obtain small ulcers on their non-pigmented side, facilitating infection in
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
43
bath challenge. Also, halibut skin may be more easily penetrable by the bacterium than
the wolffish skin. The virulence of the bacteria used for challenge should also be taken
into consideration since autogenous, and not readily comparable strains, were used for
challenge of each fish species.
In summary, halibut and spotted wolffish vaccinated with oil-emulsified vaccines
against atypical furunculosis, demonstrated a RPS of approximately 90% when challenged with homologous strains. Bath or aqueous injection vaccines failed to protect
against the disease. High antibody titres were produced in both species after vaccination
with emulsion vaccines, and antibodies were produced against A-layer, LPS and some
minor OM proteins. However, it remains to identify which antigens of the halibut and
wolffish isolates are important for protection. The heterogeneity of the marine isolates of
atypical A. salmonicida suggest that it may be necessary to include several strains in the
vaccines, and that separate vaccines may be needed for the different fish species.
Acknowledgements
This work was supported by The Norwegian Research Council and Troms Steinbit.
The authors are grateful to Ingrid Ugelstad for participating in the development of
challenge methods for halibut and wolffish, and to Alpharma, for formulating the
wolffish vaccine.
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Jones, S.R.M., Osinchuk, R.D., Mackinnon, A.M., Markham, R.J.F., 1996. Vaccination against typical
furunculosis protects against atypical Aeromonas salmonicida in salmonid and cyprinid fish. Abstract.
International Symposium on fish Vaccinology, 5–7 June 1996, Oslo, Norway, p. 79.
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Kimura, T., 1969. A new subspecies of Aeromonas salmonicida as an etiological agent of furunculosis on
‘sakuramasu’ Ž Oncorhynchus masou. and pink salmon Ž O. gorbuscha. rearing for maturity: Part 1. On the
morphological and physiological properties. Fish Pathol. 3, 34–44.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
Lund, V., Jørgensen, T., Holm, K.O., Eggset, G., 1991. Humoral immune response in Atlantic salmon, Salmo
salar L., to cellular and extracellular antigens of Aeromonas salmonicida. J. Fish Dis. 14, 443–452.
Lutwyche, P., Exner, M.M., Hancock, R.E.W., Trust, T.J., 1995. A conserved Aeromonas salmonicida porin
provides protective immunity to rainbow trout. Infect. Immun. 63, 3137–3142.
Morrissey, J.H., 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced
uniform sensitivity. Anal. Biochem. 117, 307–310.
Pyle, S.W., Cipriano, R.C., 1986. Specificity of lipopolysaccharide antigens of Aeromonas salmonicida.
Microbios Letters 31, 149–155.
Smith, I.W., 1963. The classification of Bacterium salmonicida. J. Gen. Microbiol. 33, 263–274.
Wiklund, T., Dalsgaard, I., 1998. Occurrence and significance of atypical Aeromonas salmonicida in
non-salmonid fish species: a rewiew. Dis. Aquat. Org. 32, 49–69.
www.elsevier.nlrlocateraqua-online
Vaccination of Atlantic halibut Hippoglossus
hippoglossus L., and spotted wolffish Anarhichas
minor L., against atypical Aeromonas salmonicida
Marina Ingilæ, Jan Arne Arnesen, Vera Lund ) , Guri Eggset
1
Fiskeriforskning, Norwegian Institute of Fisheries and Aquaculture, N-9291 Tromsø, Norway
Abstract
Atypical furunculosis caused by atypical Aeromonas salmonicida is an increasing problem in
commercial halibut farming, and a potential problem in farming of spotted wolffish in Norway.
Halibut, Hippoglossus hippoglossus L., and spotted wolffish, Anarhichas minor L., vaccinated
with oil-emulsified vaccines against atypical furunculosis, demonstrated a relative percent survival
ŽRPS. of approximately 90 when challenged with homologous isolates. Bath and aqueous injection
vaccines failed to protect against disease when challenged 10 weeks post-vaccination. High
antibody titres were produced in both species after vaccination with oil-emulsified vaccines, and
the major antibody response were against A-layer, LPS and some minor outer membrane ŽOM.
proteins. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Atypical Aeromonas salmonicida; Furunculosis; Vaccination; Halibut; Hippoglossus hippoglossus
L.; Spotted wolffish; Anarhichas minor L.
1. Introduction
The species Aeromonas salmonicida is, according to Bergey’s Manual of Determinative Bacteriology Ž1994., divided into three subspecies: subsp. salmonicida ŽGriffin et
al., 1953., subsp. achromogenes ŽSmith, 1963., and subsp. masoucida ŽKimura, 1969..
Other subspecies have been suggested as subsp. smithia ŽAustin et al., 1989. in addition
)
C orresponding author. T el.: q 47-77-62-90-00; fax: q 47-77-69-91-00;
vera.lund@fiskforsk.norut.no
1
Present address: Tromsø Science Park, Forskningsparken, N-9291, Tromsø, Norway.
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 2 7 9 - 3
e-m ail:
32
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
to an increasing number of reported strains that have not been assigned to any of these
subspecies. A. salmonicida subsp. salmonicida comprises a homogenous group of
strains and are referred to as typical A. salmonicida causing classical furunculosis,
while all other A. salmonicida strains are referred to as atypical and comprise a
heterogenous group causing diseases referred to as ulcer disease or atypical furunculosis
Žreviewed by Wiklund and Dalsgaard, 1998..
The number of published reports of disease outbreaks associated with atypical strains
of A. salmonicida has increased significantly during the last decade, and these isolates
have been reported from an increasing number of fish species and geographical areas
Žreviewed by Wiklund and Dalsgaard, 1998.. The virulence of atypical A. salmonicida
varies according to the host species, and variations in susceptibility to different strains of
atypical A. salmonicida are observed in both salmonids and non-salmonids. Some of the
atypical strains isolated from wild fish adversely affect salmonids and therefore may
represent a significant risk to farmed fish. At present, several non-salmonid fish species
have been introduced into farming, while other possible species are being tested for that
purpose. The susceptibility of these fish species to atypical A. salmonicida has to be
examined. Atypical furunculosis is an increasing problem in commercial halibut farming, and a potential problem in farming of spotted wolffish in Norway. Wild wolffish
caught and used as breeding stocks may be carriers of the bacterium, and outbreaks
frequently occur when the fish are stressed or when water temperature increases above
88C–108C ŽHellberg et al., 1996.. Commercial furunculosis vaccines based on A.
salmonicida subsp. salmonicida and produced for the prevention of furunculosis in
salmonids, did not give the same degree of protection against disease caused by atypical
bacterial strains in salmonids ŽGudmundsdottir and Gudmundsdottir, 1997., and probably neither against atypical furunculosis in non-salmonids. The aim of the present study
was to examine whether vaccines based on bacterial strains isolated from diseased
halibut and wolffish, may yield protective effect against atypical furunculosis in these
fish species.
2. Materials and methods
2.1. Fish
Halibut, Hippoglossus hippoglossus L. Ž; 15 g. and spotted wolffish, Anarhichas
minor L. Ž; 39 g in experiment 2 and ; 28 g in experiment 3, see Table 1. produced
from wild caught breeding stocks were used in this study. The halibut were obtained
from Lofilab, Leknes, Norway, while the wolffish were bred at the Aquaculture
Research Station in Tromsø, Norway, where the vaccination and experimental challenges were performed. The halibut were kept at 128C in a 500 l tank, and the wolffish
in raceways measuring 40 = 17 = 210 cm and 20 = 20 = 100 cm in experiments 2 and
3, respectively, both at 108C during the experiments. Prior to all handling such as
injection of vaccines or challenge bacteria or individual weighing, the fish were
anaesthetized with benzocain Ž50 mgrl water.. The different groups were spotmarked
with Alcian blue ŽPanjet..
Vaccine type
Administration
Dose
Aqueous vaccinea
Aqueous vaccinea
FIA-emulsifieda
Alpharma-emulsifieda
Unvaccinated control
Challenge method
Challenge dose
Immersion
i.p. injection
i.p. injection
i.p. injection
5=10 8 bacteriarml
2.5=10 9 bacteriarfish
2.5=10 9 bacteriarfish
2.5=10 9 bacteriarfish
a
Experiment 1 Žhalibut.
Experiment 2 Žwolffish.
Experiment 3 Žwolffish.
Fish per group
Fish per group
Fish per group
50 b
50
50
50 c
50
50
50
Bath 60 min
10 5 LFI 4050rml
50
i.p. injection
10 7 LFI 4048rfish
The halibut and the spotted wolffish vaccines contained the A. salmonicida isolates LFI 4050 and LFI 4048, respectively.
Immersion for 15 min.
c
Immersion for 45 min.
b
50
50
i.p. injection
10 4 LFI 4048rfish
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
Table 1
Experimental design
33
34
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
2.2. Bacteria
Two atypical A. salmonicida strains, LFI 4050 isolated from diseased halibut, and
LFI 4048 from diseased spotted wolffish, were used for vaccination and challenge of
halibut and spotted wolffish, respectively. They were shown to be different by biochemical tests ŽAPI 20E and API 50 CH., and by production of extracellular proteases
demonstrated in substrate gels. In addition, the strain LFI 4061 isolated from another
outbreak of atypical furunculosis in the breeding stock of spotted wolffish was used for
challenge. A typical A. salmonicida subsp. salmonicida LFI 4045 was used as a
reference strain in Western blots.
The bacteria used in the vaccines and for challenge were grown in Brain heart
infusion broth ŽBHI, Difco. containing 2% NaCl at 128C for 24 h with shaking. The
cells used in vaccines were inactivated by addition of 0.5% vol.rvol. formaldehyde
solution Ž37%. for 48 h, washed in PBS Ž20 mM phosphate, 150 mM NaCl, pH 7.4. and
resuspended in PBS before used to prepare the different vaccines ŽTable 1.. BHI agar
ŽOxoid., supplemented with 0.005% Coomassie brilliant blue, and Oxoid blood agar
base no. 2 supplemented with 2% human red blood cells and 1.5% NaCl, were used for
reisolation of bacteria from dead fish.
2.3. Vaccines and Õaccination
Three vaccination and challenge experiments were performed, one on halibut and two
on spotted wolffish. Each experiment was run in separate tanks or raceways. The
experimental design is summarised in Table 1. Four different vaccines were tested for
protection against atypical furunculosis; one immersion and three injection vaccines. The
bacterial stock suspension described above were diluted in order to obtain appropriate
cell concentrations in the different vaccines. The immersion vaccines were diluted in sea
water, the aqueous injection vaccines in PBS, and in the oil-emulsified vaccines the
bacterial suspension was diluted in PBS before emulsification in the oil adjuvant in
proportion 1:1 ŽTable 1.. The adjuvant used was either Freunds incomplete adjuvant
ŽFIA-emulsified. or the vegetable–animal oil adjuvant ŽAlpharma-emulsified. used in
the commercial furunculosis vaccines for Atlantic salmon ŽApoject-Fural vaccines,
Alpharma, Norway.. In 1998 the Apoject vaccines were replaced with the Alphaject
series which instead contains a mineral oil adjuvant. The vaccines used for the halibut
and spotted wolffish contained the A. salmonicida strains LFI 4050 and LFI 4048,
respectively. Fifty fish of each group were vaccinated either by immersion or by
intraperitoneal Ži.p.. injection of 0.1 ml of vaccines, and one group of 50 unvaccinated
fish was left as control in each experiment. In experiment 3 ŽTable 1., each fish was
weighed at the time of vaccination and challenge.
2.4. Prechallenge and challenge of fish
Challenge was performed 10 weeks post-vaccination, unless otherwise stated, with
the same A. salmonicida strains as used in the vaccines Žhomologous strains.. The
challenge method used on spotted wolffish was determined in a prechallenge experi-
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
35
ment, where groups of 10 fish were challenged with different bacterial doses by bath Ž60
min. or i.p. injection of 0.1 ml bacterial suspension. The bath and i.p. infected groups
were kept in the same raceway as an uninfected group to be infected through the water
by cohabitation.
Bath challenge, using the same bacterial concentration as had successfully been used
in previous experiments, was performed on halibut. The water level in the tank was
lowered to 50 l and the flow stopped before addition of the bacterial suspension. The
water was oxygenated during challenge.
Mortality in each group was recorded daily. The cause of death was verified by
reisolation of bacteria from kidney samples of dead fish. Cumulative mortality was
registered and relative percent survival ŽRPS. on the last day of the experiment was
calculated according to the formula: RPS s w1 y % mortality in vaccinated
groupr%mortality in control groupx = 100%..
The fish in experiment 3 surviving challenge with homologous bacterial strain,
together with a group of 50 unvaccinated, previously unchallenged fish of approximately
the same size and from the same batch, were challenged 15 weeks after the first
challenge with a heterologous A. salmonicida strain LFI 4061.
2.5. Humoral immune responses
Ten additional fish of each group in experiments 1 and 2 were vaccinated to study
specific antibody responses. The fish were sampled at the time of challenge and blood
was drained. Specific antibody responses were quantified in the sera collected, using
homologous formalin killed whole A. salmonicida cells in an enzyme-linked immunosorbent assay ŽELISA., as described by Lund et al. Ž1991.. Bound antibodies were
detected with alkaline phosphatase conjugated rabbit anti-wolffish and anti-halibut IgM
produced in the author’s laboratory.
The antibody responses were evaluated qualitatively by Western blotting, using outer
membrane ŽOM. preparations ŽFilip et al., 1973. of the two atypical A. salmonicida
strains LFI 4050 and 4048, and one typical A. salmonicida subsp. salmonicida LFI
4045 strain. The OM preparations were boiled for 5 min in 5% sodium dodecyl sulphate
ŽSDS, Sigma. with 0.2 M dithiothreitol ŽDTT, Sigma. for denaturation and reduction,
prior to separation in a 12% SDS–polyacrylamide gel ŽLaemmli, 1970.. The electrophoretic separation of the OM proteins and the transfer of the proteins ŽWestern blot.
onto a 0.45 mm nitrocellulose membrane ŽTrans-Blot w , Bio-Rad. was performed on a
Mini-PROTEAN II system ŽBio-Rad. according to the Bio-Rad Instruction Manual.
Three parallel gels were run, one was silver stained ŽMorrissey, 1981. and two were
used for Western blotting. Low Molecular Weight markers ŽPharmacia. or Kaleidoscope
markers ŽBio-Rad. were used when gels were silver stained or electroblotted, respectively.
In order to identify OM proteins reacting with the fish antibodies, the membranes
were first blocked with PBS containing 0.05% Tween 20 ŽPBS–Tween. and 5% non-fat
dry milk. Primary antisera, either from halibut or wolffish, were diluted 1:100 in
PBS–Tween with 0.5% non-fat dry milk. Secondary antibodies were the alkaline
phosphatase conjugated rabbit anti-halibut or anti-wolffish IgG mentioned above. The
36
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
membrane was incubated with the different antibodies for 60 min at room temperature
with gentle shaking, and washed 3 = 10 min with PBS–Tween between each incubation
step. Finally, the membranes were stained by adding the substrate nitroblue tetrazolium
chloride and 5-bromo-4-chloro-3-indolylphosphate ŽNBT and BCIP; Gibco BRL. in
substrate buffer Ž0.1 M Tris pH 9.5, 0.1 M NaCl and 50 mM MgCl 2 . as recommended
by the producer. The staining was stopped by washing the membrane in distilled water.
The specificity of the primary antisera had previously been verified by comparing the
reactivity of normal sera and immune sera from both halibut and wolffish to A.
salmonicida OM preparations on Western blot.
3. Results
3.1. Vaccination and challenge
The halibut were challenged by bath, as more than 50% mortality had been obtained
by such challenge Ž10 4 –10 5 cfurml. in previous trials. Prechallenge experiments
performed on wolffish showed that more that 50% of the fish died after injection
challenge with doses corresponding to 10 4 –10 7 cfurml, dependent of fish size, while
bath challenge resulted in mortalities of approximately 20% even at doses of 10 8 cfurml
Ždata not shown.. Challenge by cohabitation was not successful as the fish were not
diseased or died within a time scale of 4 weeks. Thus, injection challenge Ži.p.. was
chosen for spotted wolffish.
In experiments 1 and 2, respectively, groups of halibut and spotted wolffish were
vaccinated with the different vaccine formulations as described in Table 1, and challenged with homologous A. salmonicida strains 10 weeks later. The results are
presented as cumulative mortalities in Fig. 1. The onset of death in the bath challenged
halibut groups started 11–12 days post-challenge for all groups ŽFig. 1a.. The mortality
of the group vaccinated by immersion was similar to that of the unvaccinated group.
Compared to these, the group that obtained aqueous injection vaccine showed a lower
mortality throughout the challenge experiment. However, at the end of the experimental
period Žday 39 post-challenge., all three groups reached similar cumulative mortalities
Ž43%–47%.. On the other hand, the protection obtained in the group vaccinated with
oil-emulsified vaccine ŽFIA-emulsified. was convincing. In this group the mortality
Ž5%. was significantly lower Ž P - 0.05. than those of the other groups at the end of the
experiment, yielding RPS s 89.
The onset of death in the i.p. challenged wolffish groups occurred on day 3
post-challenge ŽFig. 1b.. All vaccinated groups and the unvaccinated control group
reached 100% mortality on day 17 post-challenge, indicating that a too high bacterial
Fig. 1. Experimental challenge of differently vaccinated and unvaccinated groups of halibut and spotted
wolffish 10 weeks post-vaccination ŽTable 1.. ŽA. The atypical A. salmonicida strain LFI 4050, from halibut,
was used in the vaccines and for bath challenge Ž10 5 rml, 60 min.. ŽB. The atypical A. salmonicida strain LFI
4048 from spotted wolffish was used in the vaccines and for i.p. challenge Ž10 7rfish..
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
37
38
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
Fig. 2. Experimental challenge of spotted wolffish 10 weeks after i.p. vaccination with A. salmonicida LFI
4048 emulsified in Alpharma’s oil adjuvant. The homologous bacterial strain was used for i.p. challenge Ž10 4
cellsrfish..
dose had been used for infection. However, on day 7, the groups vaccinated with
oil-emulsified vaccine, aqueous injection vaccine, or immersion vaccine showed cumulative mortalities of 38%, 60% and 82%, respectively, indicating the FIA-emulsified
vaccine to be the most promising vaccine candidate.
A second vaccination and challenge experiment Žexperiment 3, Table 1. was performed on wolffish, using Alpharma’s commercial oil adjuvant ŽAlpharma-emulsified.
instead of FIA. Also, a lower bacterial dose was used for this challenge. The results of
the challenge with the homologous A. salmonicida strain are shown in Fig. 2. The
unvaccinated control group started to die 5 days post-challenge, and 90% mortality was
reached on day 12. In the vaccinated group, however, only 14% cumulative mortality
was obtained 26 days post-challenge, corresponding to RPS s 90. Thus, a significant
protection against A. salmonicida was obtained in spotted wolffish when using a
vaccine containing a commercial oil adjuvant. Fifteen weeks later, 43 vaccinated fish
surviving this experiment were subjected to challenge by i.p. infection with a heteroloFig. 3. Specific antibody responses against atypical A. salmonicida in the different vaccinated groups analysed
by ELISA. ŽA. Antibody responses in halibut to strain LFI 4050. ŽB. Antibody responses in spotted wolffish to
strain LFI 4048.
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
39
40
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
gous A. salmonicida strain, isolated from moribund wild caught spotted wolffish. Also,
50 non-vaccinated and hitherto unchallenged fish were challenged as controls. All the
fish in the latter group died, but none in the vaccinated group Ždata not shown.. This
indicates that vaccines based on a single atypical A. salmonicida strain may be
protective against heterologous bacterial strains, at least those pathogenic to the same
fish species.
3.2. Humoral immune responses
The quantitative antibody responses in halibut and spotted wolffish against whole
cells of the A. salmonicida strains used in the vaccines, were determined by ELISA. By
defining the antibody titre as serum dilution yielding an optical density at 405 nm of
; 1, the oil-emulsified and aqueous injection vaccines induced titres of ) 1280 and
; 160, respectively, in both halibut and wolffish, while no specific responses were
observed in sera of immersion vaccinated fish ŽFig. 3..
Qualitative antibody responses of halibut and spotted wolffish to OM antigens of the
two atypical A. salmonicida strains used in the vaccines are shown on Western blots in
Fig. 4. The cross-reactions of these sera with OM antigens of a typical A. salmonicida
strain are also shown. Only small variations between the OM patterns of the two atypical
strains and the typical A. salmonicida strain were observed on the silver stained gel.
Antisera from halibut and spotted wolffish both reacted with the A-layer protein and
HMW LPS in the 50–60 kDa region, and some unidentified proteins with molecular
weights of 35–50 kDa in all three A. salmonicida strains. In addition the halibut
Fig. 4. Qualitative antibody responses in halibut and spotted wolffish. ŽA. Silver stained gel with OM proteins;
lane 1: low molecular weight marker, lane 2: atypical A. salmonicida LFI 4050 isolated from halibut, lane 3:
atypical A. salmonicida LFI 4048 isolated from spotted wolffish, lane 4: typical A. salmonicida subsp.
salmonicida LFI 4045. ŽB. Western blot of parallel gel as in A immunostained with halibut antiserum to A.
salmonicida LFI 4050. ŽC. Western blot of parallel gel as in A immunostained with spotted wolffish antiserum
to A. salmonicida LFI 4048.
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
41
antiserum seems to cross-react with some low molecular weight components Ž; 10 kDa.
in all three isolates.
4. Discussion
Vaccines formulated with oil adjuvants yielded better protection than aqueous
injection or bath vaccines. A protection of approximately 90% RPS could be obtained on
challenge with homologous A. salmonicida strains in both halibut and spotted wolffish
ŽFig. 1a and Fig. 2.. The efficacy of the vaccines correlated with specific antibody
responses, as the highest antibody titres were demonstrated in fish vaccinated with the
oil-emulsified vaccines. Since atypical A. salmonicida strains comprise a heterogeneous
group of bacteria, vaccinated fish should be challenged with several isolates of different
species and geographical locations, in order to determine if more than one isolate is
needed in the vaccines. In this experiment, fish shortage did not allow for challenge with
several bacterial strains. However, in experiment 3, vaccinated wolffish surviving
challenge with homologous bacterial strain, were subjected to a second challenge with a
heterologous strain. The vaccine also protected effectively against this particular strain.
The first challenge might, however, have functioned like a boost and increased the
protection of the vaccinated fish compared to the unvaccinated and previously unchallenged control fish.
The high final mortalities in all wolffish groups in experiment 2 were probably due to
the high challenge dose Ž10 7 cells per fish.. Therefore, a reduced dose Ž10 4 cells per
fish. was used in experiment 3. Even though the mortality of the unvaccinated control
group in the latter experiment also reached nearly 100%, a RPS as high as 89 was
obtained in the vaccinated group.
Vaccination with oil-emulsified vaccines seemed to reduce the growth of both halibut
and spotted wolffish. In experiments 1 and 2, an inhibition of growth was only visually
observed, since the mean weights of each vaccination group were not measured.
Therefore, in experiment 3, the mean weights of the groups were measured both at the
time of vaccination and challenge. The growth of the fish was seriously repressed as the
mean weight of the fish was 28 g when vaccinated, while at challenge 10 weeks later,
the vaccinated and unvaccinated groups were 33 and 76 g, respectively. Also, the
intestines were pale and shrunken compared to the control group, but adherances
between internal organs were rare. However, 5 months post-vaccination, the intestines of
the surviving vaccinated fish were normal and the growth rate Ž%. was the same as for
unvaccinated backup fish of the same batch.
The Alpharma-emulsified vaccine seemed to have a more serious effect on the
growth of the spotted wolffish compared to that observed in Atlantic salmon vaccinated
with Alpharma’s Apoject vaccines containing the same vegetable–animal oil adjuvant
Žinformation from Alpharma.. However, recently a vaccine containing the mineral oil
used in Alpharma’s Alphaject vaccines, was shown to have less effect on the growth of
spotted wolffish Ždata not shown., indicating that the period of growth inhibition can be
reduced.
Atypical and typical A. salmonicida strains share major cell-surface antigens such as
the A-protein layer ŽEvenberg et al., 1985. the LPS component ŽPyle and Cipriano,
42
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
1986., iron-regulated OM proteins ŽHirst and Ellis, 1994. and porins ŽLutwyche et al.,
1995., while the production of exotoxins differs ŽGudmundsdottir, 1996.. In this study,
western blots using OM preparations as antigens showed that both halibut and spotted
wolffish produced antibodies to the A-layer protein, LPS and some unidentified proteins
of homologous isolate. The antisera also cross-reacted with corresponding proteins of
typical A. salmonicida subsp. salmonicida. Little information is available about immunological protection of fish against infection with atypical A. salmonicida. Vaccination against typical furunculosis has been shown to protect against atypical furunculosis
in both salmonids and cyprinid fish ŽJones et al., 1996.. The authors of the present study
have experienced that A-layer protein on bacterial cells is an important protective
antigen in vaccines against typical furunculosis in salmonids, since no protection was
obtained with vaccines containing a bacterial strain lacking the A-layer Žunpublished
data.. Hirst and Ellis Ž1994. found that the iron-regulated membrane proteins of A.
salmonicida represented protective antigens important for successful vaccines against
both typical and atypical strains of A. salmonicida in salmon. Extracellular products
ŽECP. appeared to be important for protection against atypical A. salmonicida in carp
ŽEvenberg et al., 1988. and Atlantic salmon ŽGudmundsdottir and Magnadottir, 1997..
ECP of the subsp. achromogenes elicited better protection than whole bacteria in
Atlantic salmon, and the protection strongly correlated with the detection of antibodies
directed against the 20 kDa extracellular metallocaseinase AsaP1 in fish sera
ŽGudmundsdottir and Magnadottir, 1997..
The only commercial currently available vaccine against atypical furunculosis is an
autogenous emulsion vaccine against A. salmonicida subsp. achromogenes in salmonids
for use in Iceland where the strain is endemic ŽIceland Biojec OO, Alpharma N.W...
Both the autogenous vaccine and a commercial vaccine against classical furunculosis
ŽBiojec1500, Alpharma N.W.. evoked protection against atypical furunculosis in Atlantic salmon, the former yielding an apparently better protection than the latter
ŽGudmundsdottir and Gudmundsdottir, 1997.. On the other hand, no protection against
classical furunculosis was achieved with the autogenous vaccine. This indicate that the
two vaccines contain common antigens capable of yielding some protection against
atypical furunculosis, but insufficient to protect against classical furunculosis. However,
it remains to be tested whether the salmonid vaccine against atypical furunculosis may
protect against atypical A. salmonicida in marine fish species.
Challenge by bath or cohabitation is preferable since they mimic the infection
mechanisms in the environment. In this study, bath challenge was used on halibut, while
injection challenge Ži.p.. was used on spotted wolffish. Bath or cohabitation infection
was not successfully established in prechallenge experiments on the latter species. Both
bath and cohabitation models are commonly used, both in fresh and seawater, when
challenging commercial vaccines against typical furunculosis in Atlantic salmon. However, using A. salmonicida subsp. achromogenes, Gudmundsdottir and Gudmundsdottir
Ž1997. were only able to establish an intra muscular infection in Atlantic salmon in fresh
water. The necessity of using different challenge methods on halibut and wolffish is not
readily explained. It may reflect that farmed halibut is more susceptible to infection by
atypical A. salmonicida than farmed wolffish. Farmed halibut, inhabiting the bottom of
the tank may obtain small ulcers on their non-pigmented side, facilitating infection in
M. Ingilæ et al.r Aquaculture 183 (2000) 31–44
43
bath challenge. Also, halibut skin may be more easily penetrable by the bacterium than
the wolffish skin. The virulence of the bacteria used for challenge should also be taken
into consideration since autogenous, and not readily comparable strains, were used for
challenge of each fish species.
In summary, halibut and spotted wolffish vaccinated with oil-emulsified vaccines
against atypical furunculosis, demonstrated a RPS of approximately 90% when challenged with homologous strains. Bath or aqueous injection vaccines failed to protect
against the disease. High antibody titres were produced in both species after vaccination
with emulsion vaccines, and antibodies were produced against A-layer, LPS and some
minor OM proteins. However, it remains to identify which antigens of the halibut and
wolffish isolates are important for protection. The heterogeneity of the marine isolates of
atypical A. salmonicida suggest that it may be necessary to include several strains in the
vaccines, and that separate vaccines may be needed for the different fish species.
Acknowledgements
This work was supported by The Norwegian Research Council and Troms Steinbit.
The authors are grateful to Ingrid Ugelstad for participating in the development of
challenge methods for halibut and wolffish, and to Alpharma, for formulating the
wolffish vaccine.
References
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