Fish & Shellsh I mmunology (1996) 6, 335350

Experimental studies on the efficacy and side-effects

of intraperitoneal vaccination of Atlantic salmon
(Sal mo sal ar L.) against furunculosis
P. J . MI DTLYNG*, L. J . REI TAN AND L. SPEI LBERG
Central Veterinary Laboratory, Oslo, Norway
(Received 14 November 1995, accepted in revised form13 December 1995)
E$cacy and side-e#ects of anti-furunculosis vaccination were studied in an
experimental trial. One unadjuvanted and eight adjuvanted bacterins were
administered by intraperitoneal injection to Atlantic salmon (Salmo salar L.)
presmolts held in fresh water. Adjuvant systems represented weremineral oil,
aluminium salts, glucan and levamisole. Protection, plasma antibodies, and
side-e#ects were determined six weeks, three months, and six months after
vaccination. After six weeks, four vaccination groups were signicantly
protected compared to unvaccinated sh. Only two groups, being vaccinated
with a trivalent and a monovalent mineral oil adjuvanted vaccine, respec-
tively, were protected after three and six months. Fish fromthese two groups
were increasingly protected with time. These two groups also displayed
signicant intra-abdominal lesions. Plasma antibody levels measured against
A-layer protein and sonicated cells of Aeromonas salmonicida di#ered greatly
between groups, though all vaccinated groups mounted a signicant response
in at least one of the samplings. High antibody levels increasing with time
were found in the two long-term protected groups. At all sampling times, a
positive association between antibody levels and survival was found. I n
conclusion, only use of mineral oil adjuvanted vaccines induced durable
protective immunity against virulent waterborne furunculosis challenge.
1996 Academic Press Limited
Key words: Atlantic salmon, furunculosis, vaccination, immunity, antibody
response, side-e#ects.
I. Introduction
Furunculosis, an infectious disease of salmonid sh caused by Aeromonas
salmonicida subsp. salmonicida (Munro & Hastings, 1993), was introduced to
Norwegian sea water sh farming in 1985 (Egidius, 1987). Disease control
measures subsequently failed to prevent the spread of the disease along the
western coast of Norway. By the end of 1989, severe outbreaks among sea
water cultured Atlantic salmon (Salmo salar L.) urgently called for e#ective
disease control measures.
*Please address all correspondence to Paul J . Midtlyng, VESO VetResearch, P.O. Box 8109
Dep, N-0032 Oslo, Norway.
Present address: Regional Veterinary O$ce, Dronninggt. 15, N-3019 Drammen, Norway.
335
1050-4648/96/050335+16 $18.00 1996 Academic Press Limited
Vaccination against furunculosis has been attempted for more than
50 years, but with inconsistent results regarding protection (Hastings, 1988).
Until the beginning of this decade, few studies reporting the e$cacy of
commercially availablefurunculosis vaccines werepublished (Paterson et al.,
1985; Cardella & Eimers, 1990). More recent studies have conrmed that
signicant antibody responses and moderate protection can be obtained by
use of adjuvanted injectable bacterins, though intra-abdominal lesions were
reported as side-e#ects (Erdal & Reitan, 1992). Theduration of immunity was,
however, found to be short (Lillehaug et al., 1992). Following rapid product
development by the pharmaceutical industry, an evaluation of various
vaccines and vaccination strategies against furunculosis was initiated by
the Central Veterinary Laboratory (CVL) in Oslo in cooperation with the
Norwegian Fish Farmers Sales Organisation (FOS). The aim of the work
presentedin this paper was to study andcomparetheprotectiveimmunity and
the side-e#ects induced by a single intraperitoneal injection of di#erent
furunculosis vaccines.
II. Materials and Methods
A vaccination trial including three cohabitation challenge experiments,
accompanied by measurements of humoral antibody levels and evaluation of
intra-abdominal side-e#ects was performed at the VESO Vikan Akvavet
research facilities near Namsos, Norway.
FI SH
Approximately 4600 Atlantic salmon S1 presmolts of 32 gram average
weight, reared in fresh water, were obtained from a local hatchery, which
since 1988 had been frequently surveyed without any history of clinical or
latent furunculosis. After a short transport, the sh were randomly assigned
to ten groups of approximately 400sh each, which were placed in ten 10m
2
breglass tanks (designated vaccination tanks). Theremaining sh weresplit
into three 036m
2
tanks (designated backup tanks) for subsequent use during
the cohabitation challenge procedure. All groups were held in freshwater at
temperatures between 10and 12 C and were fed a commercial dry pellet diet
throughout the study.
VACCI NATI ON
Six monovalent (furunculosis only) and three polyvalent (combinations of
furunculosis, vibriosis and/or cold water vibriosis) vaccines wereused for the
vaccination of groups AI (Table 1). All vaccines contained formalin-
inactivated whole broth bacterins of various A. salmonicida subsp.
salmonicida strains as vaccine antigen. Except the group H bacterin, which
was grown in the presence of an iron-binding chelator, all antigens had been
produced under conventional, but recipe-specic fermentation and harvesting
conditions. Various adjuvants wereincluded in all products, except thegroup
336
P. J . MI DTLYNG ET AL.
T
a
b
l
e
1
.
I
d
e
n
t
i

c
a
t
i
o
n
o
f
A
t
l
a
n
t
i
c
s
a
l
m
o
n
p
r
e
s
m
o
l
t
g
r
o
u
p
s
i
m
m
u
n
i
s
e
d
b
y
a
s
i
n
g
l
e
i
n
t
r
a
p
e
r
i
t
o
n
e
a
l
i
n
j
e
c
t
i
o
n
w
i
t
h
v
a
r
i
o
u
s
f
u
r
u
n
c
u
l
o
s
i
s
v
a
c
c
i
n
e
s
G
r
o
u
p
V
a
c
c
i
n
e
t
y
p
e
P
r
o
d
u
c
t
i
d
e
n
t
i
t
y
M
a
n
u
f
a
c
t
u
r
e
r
A
d
j
u
v
a
n
t
s
y
s
t
e
m
I
n
j
e
c
t
e
d
v
o
l
u
m
e
A
m
o
n
o
v
a
l
e
n
t
B
i
o
j
e
c
1
5
0
0
B
i
o
m
e
d
I
n
c
.
,
U
.
S
.
A
.
m
i
n
e
r
a
l
o
i
l
0

2
m
l
B
m
o
n
o
v
a
l
e
n
t
e
x
p
e
r
i
m
e
n
t
a
l
A
q
u
a
H
e
a
l
t
h
L
t
d
,
C
a
n
a
d
a
a
l
u
m
i
n
i
u
m
p
h
o
s
p
h
a
t
e
0

1
m
l
C
b
i
v
a
l
e
n
t
1
N
o
r
v
a
x
F
U
R
P
l
u
s
N
o
r
b
i
o
A
.
S
.
,
N
o
r
w
a
y
g
l
u
c
a
n
3
0

2
m
l
D
m
o
n
o
v
a
l
e
n
t
N
o
r
v
a
x
F
U
R
N
o
r
b
i
o
A
.
S
.
,
N
o
r
w
a
y
g
l
u
c
a
n
3
0

1
m
l
E
m
o
n
o
v
a
l
e
n
t
e
x
p
e
r
i
m
e
n
t
a
l
L

v
e
n
s
k
e
m
i
s
k
e
f
a
b
r
i
k
,
D
e
n
m
a
r
k
l
e
v
a
m
i
s
o
l
e
0

1
m
l
F
t
r
i
v
a
l
e
n
t
2
B
i
o
j
e
c
1
9
0
0
B
i
o
m
e
d
I
n
c
.
,
U
.
S
.
A
.
m
i
n
e
r
a
l
o
i
l
0

2
m
l
G
t
r
i
v
a
l
e
n
t
2
e
x
p
e
r
i
m
e
n
t
a
l
A
q
u
a
c
u
l
t
u
r
e
V
a
c
c
i
n
e
s
L
t
d
,
U
.
K
.
a
l
u
m
i
n
i
u
m
h
y
d
r
o
x
i
d
e
4
0

1
m
l
H
m
o
n
o
v
a
l
e
n
t
e
x
p
e
r
i
m
e
n
t
a
l
A
q
u
a
c
u
l
t
u
r
e
V
a
c
c
i
n
e
s
L
t
d
,
U
.
K
.
n
o
n
e
0

1
m
l
I
m
o
n
o
v
a
l
e
n
t
F
u
r
o
g
e
n

A
q
u
a
H
e
a
l
t
h
L
t
d
,
C
a
n
a
d
a
a
l
u
m
i
n
i
u
m
p
h
o
s
p
h
a
t
e
0

1
m
l
J
u
n
v
a
c
c
i
n
a
t
e
d
n
o
t
a
p
p
l
i
c
a
b
l
e
(
n
.
a
.
)
n
.
a
.
n
.
a
.
n
.
a
.
1
A
e
r
o
m
o
n
a
s
s
a
l
m
o
n
i
c
i
d
a
a
n
d
V
i
b
r
i
o
s
a
l
m
o
n
i
c
i
d
a
a
n
t
i
g
e
n
s
.
2
A
e
r
o
m
o
n
a
s
s
a
l
m
o
n
i
c
i
d
a
,
V
i
b
r
i
o
s
a
l
m
o
n
i
c
i
d
a
a
n
d
V
i
b
r
i
o
a
n
g
u
i
l
l
a
r
u
m
a
n
t
i
g
e
n
s
.
3
M
a
c
r
o
g
a
r
d

,
B
i
o
t
e
c
h
-
M
a
c
k
z
y
m
a
l
A
.
S
.
,
T
r
o
m
s

.
4
A
l
h
y
d
r
o
g
e
l

,
S
u
p
e
r
p
h
o
s
A
.
S
.
,
D
e
n
m
a
r
k
.
H vaccine. The vaccines used for group A and F contained a mineral oil
adjuvant system.
After eight days of acclimatisation, the sh were anaesthetised, marked by
cold-branding, and vaccinated by a single intraperitoneal (i.p.) injection,
leaving the control sh (group J ) unvaccinated. The vaccines were adminis-
tered according to label instructions, through the ventral midline 1020mm
anterior to the pelvic ns. A benzocaine solution (50mg l
1
) was used for
anaesthesia. Self-relling syringes (Socorex 10ml) with 05mm diameter
needles wereused for injection, except for thevaccines A and F, which dueto
their viscosity were delivered using 20ml disposable syringes with 08mm
diameter needles.
ALLOCATI ON AND MONI TORI NG OF FI SH AFTER VACCI NATI ON
Post-vaccination mortality and appetite of each vaccination group was
monitored daily. After 37 days, 100 sh fromeach group were assembled into
each of three parallel 1m
2
tanks (designated challenge tanks C1C3). The
following day, the remaining sh (designated donors) were transferred to ten
036m
2
tanks awaiting blood sampling.
CHALLENGE EXPERI MENTS
Thechallengeinoculum(strain 88/09/3175CVL) was prepared as previously
described (Erdal & Reitan, 1992). LD100 was determined as 01ml out of the
highest 10-fold serial dilution giving 100% mortality in sh fromthe backup
tanks. For each challengeexperiment, 40of thesesh (designatedchallengers)
were inoculated i.p. with a double LD100 dose of viable bacteria before they
werereleasedintothechallengetank. Duringthefollowingdiseaseoutbreaks,
mortalities were recorded daily until completion of each experiment. The
specicity of mortalities was veried by identication of non-motile Gram-
negative rods producing brown di#usible pigment after cultivation fromthe
kidney on tryptic soy agar plates (Difco) for 72h at 22 C. At thecompletion of
each experiment, cumulativesurvival rates and RelativePercentageSurvival
(RPS; Amend, 1981) werecalculated. I ntroduction of challenger sh took place
six weeks (C1, rst challenge), three months (C2, second challenge) and six
months (C3, third challenge) post vaccination.
DETERMI NATI ON OF HUMORAL ANTI BODY LEVELS
Simultaneous to the start of each challenge experiment, 25 sh fromeach
donor tank were netted, stunned by a blow to the head, and bled into
heparinised tubes fromthe caudal vein. The blood samples were kept on ice,
and, within four hours, centrifuged for 10 min. The plasma was stored at
20 C until further analysis. The enzyme-linked immunosorbent assay
(ELI SA) for determination of antibody activity to A. salmonicida antigens
was performed as described previously (Erdal & Reitan, 1992), with minor
modications. A sonicate of strain CVL 86/09/3173 A. salmonicida subsp.
salmonicida cells containing 3g protein ml
1
, or puried A-layer protein
(Phipps et al., 1983) fromthis strain at 2g protein ml
1
served as primary
338
P. J . MI DTLYNG ET AL.
antigen. Polystyrene plates were coated with 100l antigen solution (005M
carbonate bu#er; pH 96) per well. After adding plasma, the plates were
incubated overnight at +4 C. A monoclonal mouseantibody against rainbow
trout immunoglobulin (clone 4C10; Thuvander et al., 1990) was used as
secondary antibody, followed by a sheep anti-mouse-I g conjugated to per-
oxidase (Amersham, Buckinghamshire, U.K.); both incubated for 45 min at
37 C. Thesubstrateused was tetramethylbenzidine(TMB; Merck, Darmstadt,
Germany). A pooled positive plasma, obtained from Atlantic salmon vacci-
nated twicei.p. with vaccineI was run on each plateas reference. Theresults
wereexpressedas theratiobetween themean absorbances of duplicatesample
and reference pool wells, read at 450nm. After initial calibration, a plasma
dilution of 1:50was selected for the completion of the antibody analyses. Due
to high ELI SA readings, group A and group F samples were additionally run
at 1:100 and 1:1000 dilutions.
EVALUATI ON OF I NTRA-ABDOMI NAL LESI ONS
At each bloodsampling, veblooddonorsfromeach vaccination groupwere
randomly selected and shipped on ice to the laboratory for autopsy the
following day. Each sh was given a score from zero to six reecting the
severity of lesions according to the visual appearance of the body cavity
(Table 2). The evaluation was performed by one investigator, who was left
blind regarding the group identity of the sh.
STATI STI CAL ANALYSI S
Survival data fromthe challenge experiments were tested by log rank tests
of the calculated Kaplan Meier survival function, using SPSS statistical
software(SPSS ReferenceGuide, Chapter 10). Antibody ELI SA readings were
compared by analysis of variance, using Epi I nfo statistical software (Dean
et al., 1990). Where these data were not normally distributed or variances
were inhomogenous, the reported P-values refer to non-parametric tests
(KruskalWallis one-way analysis of variance or MannWhitney U test).
Autopsy results wereanalysed using Fishers exact test, and Spearmans rank
correlation was calculated for analysis of association between survival and
antibody data (SAS

Users guide, 4th edition, chapter 19). A conservative

signicance level (P<001) was applied where the analysis included pairwise
testing between multiple groups.
III. Results
POST-VACCI NATI ON MORTALI TI ES AND APPETI TE
Three sh died during the rst 24h after vaccination, and 16 sh died
during the following 36 days, being non-signicantly distributed between
vaccination groups. Attempts to isolate A. salmonicida from the kidney of
thesesh werenegative. After formation of thechallengepopulations, a total
of four sh from tank C2, and nine sh from tank C3 su#ered non-specic
mortality prior to initiation of challenge. Reduced appetite was seen in all
VACCI NATI ON AGAI NST FURUNCULOSI S
339
T
a
b
l
e
2
.
C
l
a
s
s
i

c
a
t
i
o
n
s
c
h
e
m
e
f
o
r
i
n
t
r
a
-
a
b
d
o
m
i
n
a
l
l
e
s
i
o
n
s
s
e
e
n
a
f
t
e
r
i
n
t
r
a
p
e
r
i
t
o
n
e
a
l
v
a
c
c
i
n
a
t
i
o
n
o
f
A
t
l
a
n
t
i
c
s
a
l
m
o
n
p
r
e
s
m
o
l
t
s
w
i
t
h
v
a
r
i
o
u
s
f
u
r
u
n
c
u
l
o
s
i
s
v
a
c
c
i
n
e
s
S
c
o
r
e
V
i
s
u
a
l
a
p
p
e
a
r
a
n
c
e
o
f
a
b
d
o
m
i
n
a
l
c
a
v
i
t
y
S
e
v
e
r
i
t
y
o
f
l
e
s
i
o
n
s
0
N
o
v
i
s
i
b
l
e
l
e
s
i
o
n
s
N
o
n
e
1
V
e
r
y
s
l
i
g
h
t
a
d
h
e
s
i
o
n
s
,
m
o
s
t
f
r
e
q
u
e
n
t
l
y
l
o
c
a
l
i
s
e
d
c
l
o
s
e
t
o
t
h
e
i
n
j
e
c
t
i
o
n
s
i
t
e
.
N
o
o
r
m
i
n
o
r
o
p
a
q
u
i
t
y
o
f
p
e
r
i
t
o
n
e
u
m
a
f
t
e
r
U
n
l
i
k
e
l
y
t
o
b
e
n
o
t
i
c
e
d
b
y
l
a
y
m
e
n
d
u
r
i
n
g
e
v
i
s
c
e
r
a
t
i
o
n
e
v
i
s
c
e
r
a
t
i
o
n
2
M
i
n
o
r
a
d
h
e
s
i
o
n
s
,
w
h
i
c
h
m
a
y
c
o
n
n
e
c
t
c
o
l
o
n
,
s
p
l
e
e
n
o
r
c
a
u
d
a
l
p
y
l
o
r
i
c
c
a
e
c
a
t
o
t
h
e
O
n
l
y
o
p
a
s
i
c
i
t
y
o
f
p
e
r
i
t
o
n
e
u
m
r
e
m
a
i
n
i
n
g
a
b
d
o
m
i
n
a
l
w
a
l
l
.
M
a
y
b
e
n
o
t
i
c
e
d
b
y
l
a
y
m
e
n
d
u
r
i
n
g
e
v
i
s
c
e
r
a
t
i
o
n
a
f
t
e
r
m
a
n
u
a
l
l
y
d
i
s
c
o
n
n
e
c
t
i
n
g
t
h
e
a
d
h
e
s
i
o
n
s
3
M
o
d
e
r
a
t
e
a
d
h
e
s
i
o
n
s
i
n
c
l
u
d
i
n
g
m
o
r
e
c
r
a
n
i
a
l
p
a
r
t
s
o
f
t
h
e
a
b
d
o
m
i
n
a
l
c
a
v
i
t
y
,
p
a
r
t
l
y
M
i
n
o
r
v
i
s
i
b
l
e
l
e
s
i
o
n
s
a
f
t
e
r
e
v
i
s
c
e
r
a
t
i
o
n
,
i
n
v
o
l
v
i
n
g
p
y
l
o
r
i
c
c
a
e
c
a
,
t
h
e
l
i
v
e
r
o
r
v
e
n
t
r
i
c
l
e
,
c
o
n
n
e
c
t
i
n
g
t
h
e
m
t
o
t
h
e
a
b
d
o
m
i
n
a
l
w
h
i
c
h
m
a
y
b
e
r
e
m
o
v
e
d
m
a
n
u
a
l
l
y
w
a
l
l
.
M
a
y
b
e
n
o
t
i
c
e
d
b
y
l
a
y
m
e
n
d
u
r
i
n
g
e
v
i
s
c
e
r
a
t
i
o
n
4
M
a
j
o
r
a
d
h
e
s
i
o
n
s
w
i
t
h
g
r
a
n
u
l
o
m
a
,
e
x
t
e
n
s
i
v
e
l
y
i
n
t
e
r
c
o
n
n
e
c
t
i
n
g
i
n
t
e
r
n
a
l
o
r
g
a
n
s
,
M
o
d
e
r
a
t
e
l
e
s
i
o
n
s
w
h
i
c
h
m
a
y
b
e
h
a
r
d
t
o
w
h
i
c
h
t
h
e
r
e
b
y
a
p
p
e
a
r
a
s
o
n
e
u
n
i
t
.
L
i
k
e
l
y
t
o
b
e
n
o
t
i
c
e
d
b
y
l
a
y
m
e
n
d
u
r
i
n
g
r
e
m
o
v
e
m
a
n
u
a
l
l
y
e
v
i
s
c
e
r
a
t
i
o
n
5
E
x
t
e
n
s
i
v
e
l
e
s
i
o
n
s
a
#
e
c
t
i
n
g
n
e
a
r
l
y
e
v
e
r
y
i
n
t
e
r
n
a
l
o
r
g
a
n
i
n
t
h
e
a
b
d
o
m
i
n
a
l
c
a
v
i
t
y
.
L
e
a
v
i
n
g
v
i
s
i
b
l
e
d
a
m
a
g
e
t
o
t
h
e
c
a
r
c
a
s
s
a
f
t
e
r
I
n
l
a
r
g
e
a
r
e
a
s
,
t
h
e
p
e
r
i
t
o
n
e
u
m
i
s
t
h
i
c
k
e
n
e
d
a
n
d
o
p
a
q
u
e
,
a
n
d
t
h
e

l
l
e
t
m
a
y
c
a
r
r
y
e
v
i
s
c
e
r
a
t
i
o
n
a
n
d
r
e
m
o
v
a
l
o
f
l
e
s
i
o
n
s
f
o
c
a
l
,
p
r
o
m
i
n
e
n
t
a
n
d
/
o
r
h
e
a
v
i
l
y
p
i
g
m
e
n
t
e
d
l
e
s
i
o
n
s
o
r
g
r
a
n
u
l
o
m
a
s
6
E
v
e
n
m
o
r
e
p
r
o
n
o
u
n
c
e
d
t
h
a
n
5
,
o
f
t
e
n
w
i
t
h
c
o
n
s
i
d
e
r
a
b
l
e
a
m
o
u
n
t
s
o
f
m
e
l
a
n
i
n
.
L
e
a
v
i
n
g
m
a
j
o
r
d
a
m
a
g
e
t
o
t
h
e
c
a
r
c
a
s
s
V
i
s
c
e
r
a
u
n
r
e
m
o
v
a
b
l
e
w
i
t
h
o
u
t
d
a
m
a
g
e
t
o

l
l
e
t
i
n
t
e
g
r
i
t
y
groups of sh for approximately 3 weeks after vaccination, without apparent
di#erences between groups.
CHALLENGE EXPERI MENTS
Theprogress of thethreechallengeexperiments was uniform, and mortality
curves fromtank C2aredisplayedas an example(Fig. 1). Mortality amongi.p.
infected challengers started after 35 days, inducing a furunculosis epizootic
among the study sh starting 79 days later. At the end of the challenge
experiments, cumulative mortalities were higher than 90% (C1, C2) or
approximately 60% (C3) in unvaccinated controls, respectively (Table 3).
When challenged six weeks after vaccination, the survival of group C, D,
E and F sh was signicantly improved compared to the unvaccinated sh
(P<0001). Relative protection was, however, below 40% (Table 3). When
challenged after three months, only group A and F sh survived signi-
cantly longer than the control sh (P<<0001), the relative protection being
low in group A but moderate (58%) in group F sh. These two groups were
signicantly protected also during the third challenge, six months after
vaccination (P<0005). Among the protected groups, the highest level of
protection was consistently seen in group F, increasing from 34% RPS
during the rst challenge to 87% RPS during the third challenge exper-
iment. This vaccine consistently induced superior protection compared to its
monovalent counterpart group A (P<0001), and indications for a similar
e#ect were seen when comparing the bivalent vaccine group (C) v. the
corresponding monovalent vaccine group (D) during the rst challenge
experiment (P =0038). The improved survival initially seen in the groups C,
D and E was not found during the second and third challenge. I n group A,
onset of immunity was found after three months. Similar to group F, this
group showed increasing relative survival pattern with time.
25
100
0
0
Day
C
u
m
u
l
a
t
i
v
e

m
o
r
t
a
l
i
t
y

i
n

%
80
60
40
20
5 10 15 20
Fig. 1. Mortality of Atlantic salmon presmolts during a cohabitation challenge
experiment initiated three months after intraperitoneal vaccination against furun-
culosis (tank C2). Ten groups of approximately 100 sh each were tested. Group
designations are given in Table 1. Only data from protected groups (P<001) are
displayed. , A; , F; , J ; , challengers.
VACCI NATI ON AGAI NST FURUNCULOSI S
341
T
a
b
l
e
3
.
C
u
m
u
l
a
t
i
v
e
m
o
r
t
a
l
i
t
y
r
a
t
e
s
a
n
d
r
e
l
a
t
i
v
e
p
e
r
c
e
n
t
a
g
e
s
u
r
v
i
v
a
l
(
R
P
S
)
o
f
t
e
n
g
r
o
u
p
s
o
f
A
t
l
a
n
t
i
c
s
a
l
m
o
n
p
r
e
s
m
o
l
t
s
v
a
c
c
i
n
a
t
e
d
w
i
t
h
d
i
#
e
r
e
n
t
v
a
c
c
i
n
e
s
,
a
s
d
e
t
e
r
m
i
n
e
d
a
t
t
h
e

n
a
l
d
a
y
o
f
t
h
r
e
e
c
h
a
l
l
e
n
g
e
e
x
p
e
r
i
m
e
n
t
s
w
i
t
h
A
e
r
o
m
o
n
a
s
s
a
l
m
o
n
i
c
i
d
a
3
V
a
c
c
i
n
e
V
a
c
c
i
n
e
t
y
p
e
F
i
r
s
t
c
h
a
l
l
e
n
g
e
(
a
f
t
e
r
s
i
x
w
e
e
k
s
)
S
e
c
o
n
d
c
h
a
l
l
e
n
g
e
(
a
f
t
e
r
t
h
r
e
e
m
o
n
t
h
s
)
T
h
i
r
d
c
h
a
l
l
e
n
g
e
(
a
f
t
e
r
s
i
x
m
o
n
t
h
s
)
D
e
a
d
S
u
r
v
i
v
e
d
R
P
S
D
e
a
d
S
u
r
v
i
v
e
d
R
P
S
D
e
a
d
S
u
r
v
i
v
e
d
R
P
S
A
B
i
o
j
e
c
1
5
0
0
m
o
n
o
v
a
l
e
n
t
9
1
9
2
7
5
2
2
1
9
*
4
7
5
2
2
5
*
B
e
x
p
e
r
i
m
e
n
t
a
l
m
o
n
o
v
a
l
e
n
t
8
6
1
4
7
8
9
1
1
7
5
6
4
3
1
0
C
N
o
r
v
a
x
F
U
R
p
l
u
s
b
i
v
a
l
e
n
t
7
7
2
3
1
7
*
8
8
1
1
7
6
1
3
6
0
D
N
o
r
v
a
x
F
U
R
m
o
n
o
v
a
l
e
n
t
8
6
1
5
8
*
9
1
8
4
4
8
4
3
1
6
E
e
x
p
e
r
i
m
e
n
t
a
l
m
o
n
o
v
a
l
e
n
t
8
2
1
8
1
2
*
9
4
6
2
8
2
3
2

1
4
F
B
i
o
j
e
c
1
9
0
0
t
r
i
v
a
l
e
n
t
6
1
3
9
3
4
*
4
0
5
9
5
8
*
8
9
0
8
7
*
G
e
x
p
e
r
i
m
e
n
t
a
l
t
r
i
v
a
l
e
n
t
9
4
7
0
9
5
7
3
5
9
3
4

1
H
e
x
p
e
r
i
m
e
n
t
a
l
m
o
n
o
v
a
l
e
n
t
9
8
2

5
9
2
6
2
6
8
3
2

8
I
F
u
r
o
g
e
n

m
o
n
o
v
a
l
e
n
t
8
9
1
2
5
9
4
6
2
6
8
3
4

6
J
u
n
v
a
c
c
i
n
a
t
e
d
9
3
7
9
6
4
6
1
3
6
3
*
S
i
g
n
i

c
a
n
t
(
P
<
0

0
1
)
v
.
u
n
v
a
c
c
i
n
a
t
e
d
c
o
n
t
r
o
l
s
.
HUMORAL I MMUNE RESPONSES
ELI SA absorbances of the positive pool sample were uniform, with average
values of 102 (against sonicated cells) and 076 (A-layer), respectively, when
diluted 1:50.
Elevated antibody levels against one or both antigen preparations of A.
salmonicida (A-layer protein and sonicated cells) were found in all groups of
vaccinated sh after six weeks (Fig. 2). Except for group A and F, where
readings were equal to or exceeded pool values, plasma antibodies were
strongly reduced, and in some groups completely absent after three and six
months. I n contrast to all other groups, the antibody level of group A and
group F increased towards the end of the study, nally reaching levels equal
toor abovethereferenceplasmasamplewhich wasusedasapositivestandard
in the assay.
Signicantly higher antibody levels against A-layer, compared to unvacci-
nated sh, were found only in sh from group A (P<<0001), group F
(P<<0001) and group I (P<0001) six months after vaccination [Fig. 2(a)].
Anti-A-layer antibodies were consistently higher in group F (trivalent vacci-
nated sh) compared to group A (monovalent vaccinated sh) (P<0001). The
level of antibodies against sonicated cells showed a similar pattern [Fig. 2(b)].
Against this antigen preparation, all but group G and group H sh remained
signicantly responsive after six months. Comparing the antibody levels
against sonicated A. salmonicida cells in plasma diluted 1:1000, signicantly
higher levels were found in group F than in group A sh after six weeks
(P =001), but not after three and six months.
A signicant positive correlation (P<005) between the survival rates and
the corresponding median antibody levels of each group was found at all the
challenge times. Of the two humoral parameters, A-layer antibodies consist-
entlyyieldedthehighest correlation coe$cientswhen plottedagainst survival
(Fig. 3).
I NTRA-ABDOMI NAL LESI ONS
I ntra-abdominal lesions werevisiblein oneor moresh fromall vaccinated
groups at all three sampling times. The highest scores of these side-e#ects
wererecorded in group A and group F sh after six months (Fig. 4). Thesetwo
mineral oil adjuvant vaccine groups displayed the highest scores also in the
rst two samplings.
IV. Discussion
The results provide rm evidence that the use of mineral oil adjuvanted
furunculosis vaccines induced a long-lasting protective immunity. Unlike the
other groups, signicant protection and high antibody levels were found in
salmon vaccinatedwith monovalent (groupA) andtrivalent (groupF) vaccine
three and six months after vaccination. These ndings are in agreement with
early reports on experimental furunculosis vaccination of salmonids showing
that signicant protection against furunculosis can be obtained by use of oil
adjuvanted A. salmonicida bacterins (Krantz et al., 1963; Krantz et al., 1964;
VACCI NATI ON AGAI NST FURUNCULOSI S
343
Paterson & Fryer, 1974; Cipriano & Pyle, 1985). I n the mineral oil vaccine
groups, antibody responses as well as protection increased during the course
of the study, whereas a pattern of decreasing immunity was observed in all
other groups. Thesedi#erences in level and development of immunity suggest
0
A
Vacci nati on group
J
1.0
1.2
0.8
0.6
0.4
0.2
B C D E F G H I
0
A
O
D

s
a
m
p
l
e
/
O
D

p
o
s
.

r
e
f
e
r
e
n
c
e

s
a
m
p
l
e
J
1.0
1.4
0.8
0.6
0.4
0.2
B C D E F G H I
1.2
(a)
(b)
*
*
*
*
*
* * * * **
*
*
*
*
Fig. 2. Plasma antibody levels measured against A-layer protein (a), and sonicated A.
salmonicida cells (b) in Atlantic salmon vaccinated with ninedi#erent furunculosis
vaccines (AH), and in unvaccinated sh (J ). ELI SA absorbances were read at
450nmin samples diluted1:50(N=25). Median and75-percentiles of 25samples from
each group drawn at three intervals after vaccination are shown. , Six weeks; ,
three months; , six months. *=non-signicant (P>001) v. unvaccinated controls.
344
P. J . MI DTLYNG ET AL.
that useof mineral oil adjuvant vaccines havemajor advantages for immuno-
prophylaxis of salmon compared to water-based products. The results seen in
group D and I sh agree with antibody response studies with products
containingnon-oil adjuvants (Erdal & Reitan, 1992; Rrstadet al., 1993; Aakre
et al., 1994).
1.4
100
0
0
Sampl e OD/OD pos. reference sampl e
1
80
60
40
20
0.2 0.4 0.6 0.8 1.2
(c)
1.4
100
0
0
%

s
u
r
v
i
v
a
l

a
f
t
e
r

c
h
a
l
l
e
n
g
e
1
80
60
40
20
0.2 0.4 0.6 0.8 1.2
(b)
1.4
100
0
0 1
80
60
40
20
0.2 0.4 0.6 0.8 1.2
(a)
Fig. 3. Association between median relative antibody response to A-layer and cumu-
lative survival during experimental challenge in nine groups of Atlantic salmon
presmolts. Data were obtained six weeks (a), three months (b), and six months (c)
after vaccination against furunculosis. Signicantly protected groups areshown by
solidsymbols. Spearmans rank correlation coe$cients (asymptotic standarderrors)
were 082 (011), 071 (024) and 065 (022), respectively.
VACCI NATI ON AGAI NST FURUNCULOSI S
345
I t is believed that the superior survival of group A and group F vaccinates
is mainly attributable to the formulation of the vaccines, in particular to the
inclusion of a potent adjuvant system with the Aeromonas bacterin. The
immuno-enhancing e#ects of mineral oil adjuvants have been known for
nearly 50 years (Freund, 1947), and have been repeatedly demonstrated in
salmonid sh (Krantz et al., 1964; Udey & Fryer, 1978; Palmer & Smith, 1980;
Olivier et al., 1985a). Their mode of action is thought to be repository,
stimulating the production of antibodies over long periods of time. Oil
emulsion vaccines are therefore regarded as strong stimulators of humoral,
and weaker stimulators of cellular immunity (Vanselow, 1987). Theslowonset
of immunity and the high antibody levels observed in the present studies are
in agreement with features of oil adjuvants, as is thedemonstration of antigen
retention and high enzymatic activities in the spleen of group F sh fromthe
present study reportedby Press et al. (1995). Such a pattern of slowdeveloping
immunity also implies, however, that no signicant protection may be
revealed if a low number of sh are tested within a few weeks after vaccina-
tion. This may be one important reason why some authors have found no or
only minor benecial e#ects from mineral oil adjuvants in furunculosis
vaccines (Cipriano, 1982; McCarthy et al., 1983; Adams et al., 1988).
I n the present data, antibody levels and protection were consistently
positively associated. This ndingappears to contradict conclusions drawn by
others (Michel, 1979; Michel & Faivre, 1982; Cipriano, 1983; Olivier et al.,
1985a). An explanation for this disagreement cannot be o#ered here, as the
cited papers fail to report results from statistical analysis of association.
Whether the associations observed in our data are causal or coincidental, or
whether associations arepresent on individual sh level cannot beclariedby
the present study. The distribution of data further prevents the raising of a
rm hypothesis regarding which association model (linear or threshold) is
Fig. 4. Range and mean score of intra-abdominal lesions recorded six months after
intraperitoneal vaccination of Atlantic salmon presmolts with di#erent vaccines
against furunculosis. N=5 sh per group; overall P<005.
346
P. J . MI DTLYNG ET AL.
more appropriate. Nevertheless, the results suggest that monitoring humoral
antibody levels or responses against A. salmonicida antigens may have a
potential for predicting furunculosis vaccine e$cacy. The feasibility of anti-
body assays during development of new furunculosis vaccines, and for the
replacement of challenge experiments during batch potency testing should
therefore be subject to further investigations.
Remarkably, antibodies against A-layer correlated stronger with protection
than antibodies against sonicated cells. A signicant positive correlation
between the genetically determined responsiveness to A-layer after vaccina-
tion and the natural resistance to furunculosis challenge in unvaccinated
families of Atlantic salmon has been demonstrated earlier (Lund et al., 1995).
I n contrast to conclusions drawn by Trust (1986), the present data therefore
suggest that the role of this antigen in protection deserves further attention.
Olivier et al. (1985b) claimed that theimmunity conferred by oil adjuvanted
furunculosis vaccines is of a non-specic nature, and this view has later been
supported by Adams et al. (1988). We agree that non-specic immunostimula-
tion mayhavecontributedtotheprotection observedin thepresent study. The
superior specic humoral immune responses induced by the mineral oil
adjuvanted vaccines are nevertheless underscoring the specic component of
the response, and that a true adjuvant e#ect according to the denition
o#ered by Ramon (1926) was achieved. As both mineral oil adjuvanted
vaccines included in our studies contained equal amounts of the same
adjuvant system (Goodrich, pers. comm.), di#erences in antibody responses
and protection emphasise the antigenic composition of the products over
adjuvant factors.
The superior immunity found in sh vaccinated with polyvalent vaccine
products (groups F and C) compared to their corresponding monovalent
parallels (groups A andD, respectively), was surprising. Theseresults support
thendingsof Strmsheim(1993), whofoundaco-adjuvant e#ect of an antigen
mixture containing V. anguillarum antigens when assessing the humoral
response to A. salmonicida A-layer. Our ndings suggest that the protective
e#ect of furunculosis vaccines can be enhanced by the composition of
polyvalent products, and should encourage further research on this aspect of
vaccine development, a position which has been advocated earlier (Amend &
J ohnson, 1984). Previous reports of cross-protection against furunculosis after
vaccination against vibriosis (Adams et al., 1988; Norqvist et al., 1989) suggest
that specic as well as non-specic mechanisms of immunity might contribute
to the observed e#ect.
I n contrast to previous reports (Paterson et al., 1985; Erdal & Reitan, 1992;
Lillehaug et al., 1992; Rrstad et al., 1993; Aakreet al., 1994), vaccination with
aluminium salt or glucan-adjuvanted bacterins failed to yield protective
immunity in our experiments. This disagreement may in part be caused by
the infectious pressure employed here, which gave very high control
mortalities (up to 90%). The severity of the challenge may also be the reason
why the results obtained from group H sh in our study were rather
disappointing, asany protectivee#ectsof A. salmonicidaantigen grown under
iron-limited conditions, as reported by Hirst & Ellis (1994) could not be
conrmed.
VACCI NATI ON AGAI NST FURUNCULOSI S
347
The absence of any signicant immediate or delayed post vaccination
mortalities conrms that intraperitoneal vaccination of salmonids is safe,
when skilfully performed (Larsen, 1987; Lillehaug, 1989). I n spite of the
presence of injection-site lesions reduced appetite or growth, aberrant behav-
iour of the vaccinated sh was not recorded beyond three weeks after
vaccination. Theintraperitoneal lesions found in themajority of group A and
group F sh were clearly visible to the naked eye, their macroscopic and
microscopic appearance being similar to or in excess of the lesions described
by Lillehaug et al. (1992). To what degree these lesions will remain visible
through to market size of the sh, remains to be claried.
I n conclusion, the results obtained in the present study demonstrate that
intraperitoneal vaccination using adjuvanted bacterins is e#ective against
waterbornefurunculosis challenge, andthat theuseof mineral oil adjuvanted
vaccines will protect salmon e#ectively against diseasefor at least six months.
The presence of visible injection-site lesions was not associated with mortali-
ties nor major signs of discomfort. Thus, the results obtained in this study
provide scientic evidence for the feasibility of furunculosis vaccination in
salmonid sh farming, and that a long awaited milestone (Munro, 1984) has
now been reached.
Thanks are due to the Norwegian Fish Farming Sales Organisation (FOS) for their
cooperation during the initiation of the project during which these studies were
performed. We are grateful for the contribution of the vaccine manufacturers Biomed
I nc., Apotekernes Laboratorium A.S., Norbio A.S., Lvens Kemiske Fabrik and
Aquaculture Vaccines, Ltd, who kindly submitted vaccines for testing and, together
with the Central Veterinary Laboratory and VESO A.S. (the Norwegian animal
vaccinedistributor), supported thenancial basis of theproject. Thanks aredueto Dr
AnneRamstad of VESO Vikan AkvaVet research station for supervising and conduct-
ing experimental work; to Mrs Karen Bkken Soleimfor accomplishments of labora-
toryanalyses; andtoDr J on I ngeErdal for hiscontribution tothedesign andinitiation
of the trials.
References
Aakre, R., Wergeland, H. I ., Aasjord, P. M. & Endresen, C. (1994). Enhanced antibody
response in Atlantic salmon (Salmo salar L.) to Aeromonas salmonicida cell
wall antigens using a bacterin containing -1,3-M-Glucan as adjuvant. Fish &
Shellsh I mmunology 4, 4761.
Adams, A., Auchinachie, N., Bundy, A., Tatner, M. F. & Horne, M. T. (1988). The
potency of adjuvanted injected vaccines in rainbow trout (Salmo gairdneri
Richardson) and bath vaccines in Atlantic salmon (Salmo salar L.) against
furunculosis. Aquaculture69, 1526.
Amend, D. F. (1981). Potency testingof sh vaccines. I n Fish Biologics: Serodiagnostics
and Vaccines (W. Hennessen, ed.) pp. 447454. Basel: S. Karger.
Amend, D. F. & J ohnson, K. A. (1984). Evidence of lack of competition among various
combinations of Vibrio anguillarum, Yersinia ruckeri, Aeromonas salmonicida
and Renibacterium salmoninarum bacterins when administered to salmonid
shes. J ournal of Fish Diseases 7, 293299.
Cardella, M. A. & Eimers, M. E. (1990). Safety and potency testingof federally licensed
sh bacterins. J ournal of Aquatic Animal Health 2, 4955.
Cipriano, R. C. (1982). I mmunogenic potential of growth products extracted from
cultures of Aeromonas salmonicida for brook trout (Salvelinus fontinalis).
Canadian J ournal of Fisheries and Aquatic Sciences 39, 15121518.
348
P. J . MI DTLYNG ET AL.
Cipriano, R. C. (1983). Resistance of salmonids to furunculosis: relation between
agglutinins and neutralizing activities. Transactions of theAmerican Fisheries
Society 112, 9599.
Cipriano, R. C. & Pyle, S. W. (1985). Adjuvant-dependent immunity and the agglutinin
response of shes against Aeromonas salmonicida, cause of furunculosis.
Canadian J ournal of Fisheries and Aquatic Sciences 42, 12901295.
Dean, A. G., Dean, J . A., Burton, A. H. & Dicker, R. C. (1990). Epi I nfo, Version 5: a
Word Processing, Database, and Statistics Program for Epidemiology on
Microcomputers. Stone Mountain, Georgia, U.S.A.: USD, I nc.
Egidius, E. (1987). I mport of furunculosis to Norway with Atlantic salmon smolts
from Scotland. I nternational Council for the Exploration of the Sea (I CES),
Mariculture Committee C.M. 1987/F:8.
Erdal, J . I . & Retain, L. J . (1992). I mmune response and protective immunity after
vaccination of Atlantic salmon (Salmo salar L.) against furunculosis. Fish &
Shellsh I mmunology 2, 99108.
Freund, J . (1947). Someaspects of activeimmunization. Annual Reviewof Microbiology
1, 291308.
Hastings, T. S. (1988). Furunculosis vaccines. I n Fish Vaccination (A. E. Ellis, ed.)
pp. 93111. London: Academic Press.
Hirst, I . D. & Ellis, A. E. (1994). I ron-regulated outer membraneproteins of Aeromonas
salmonicida are important protective antigens in Atlantic salmon against
furunculosis. Fish & Shellsh I mmunology 4, 2945.
Krantz, G. E., Reddecli#, J . M. & Heist, C. E. (1963). Development of antibodies against
Aeromonas salmonicida in trout. J ournal of I mmunology 91, 757760.
Krantz, G. E., Reddecli#, J . M. & Heist, C. E. (1964). I mmune response of trout to
Aeromonas salmonicida. Part I . Development of agglutinating antibodies and
protective immunity. TheProgressiveFish-Culturist 26, 310.
Larsen, J . L. (1987). (Field trial with combined vaccines for trout in marine sh
farms) Feltforsg med kombinationsvacciner til rreder i havbrug. Dansk
Veterinrtidsskrift 70, 382385 (in Danish).
Lillehaug, A. (1989). A survey on di#erent procedures used for vaccinating salmonids
against vibriosis in Norwegian sh farming. Aquaculture83, 217226.
Lillehaug, A., Lunder, T. & Poppe, T. T. (1992). Fieldtestingof adjuvantedfurunculosis
vaccines in Atlantic salmon, Salmo salar L. J ournal of Fish Diseases 15,
485496.
Lund, T., Chiayvareesajja, J ., Larsen, H. J . S. & Red, K. H. (1995). Antibody response
after immunization as a potential indirect marker for improved resistance
against furunculosis. Fish & Shellsh I mmunology 5, 109119.
McCarthy, D. H., Amend, D. F., J ohnson, K. A. & Bloom, J . V. (1983). Aeromonas
salmonicida: determination of an antigen associated with protective
immunity and evaluation of an experimental bacterin. J ournal of Fish Diseases
6, 155174.
Michel, C. (1979). Furunculosis of salmonids; vaccination attempts in rainbow trout
(Salmo gairdneri) by formalin-killed germs. Annales de Recherche Veterinaire
10, 3340.
Michel, C. & Faivre, B. (1982). Occurrenceandsignicanceof agglutinatingantibodies
in experimental furunculosis of rainbow trout, Salmo gairdneri Richardson.
J ournal of Fish Diseases 5, 429432.
Munro, A. L. S. (1984). A furunculosis vaccineillusion or achievable objective. I n
Symposium on Fish Vaccination (P. Kinkelin, ed.) pp. 97120. Paris: O$ce
I nternational des Epizooties.
Munro, A. L. S. & Hastings, T. S. (1993). Furunculosis. I n Bacterial Diseases of Fish (V.
I nglis, R. J . Roberts & N. R. Bromage, eds) pp. 122142. Oxford: Blackwell
Scientic Publications.
Norqvist, A., Hagstrom, A. & Wolf-Watz, H. (1989). Protection of rainbowtrout against
vibriosis and furunculosis by the use of attenuated strains of Vibrio
anguillarum. Applied and Environmental Microbiology 55, 14001405.
VACCI NATI ON AGAI NST FURUNCULOSI S
349
Olivier, G., Evelyn, T. P. T. & Lallier, R. (1985a). I mmunogenicity of vaccines froma
virulent and an avirulent strain of Aeromonas salmonicida. J ournal of Fish
Diseases 8, 4355.
Olivier, G., Evelyn, T. P. T. & Lallier, R. (1985b). I mmunity to Aeromonas salmonicida
in coho salmon (Onchorynchus kisutch) induced by modied Freunds complete
adjuvant: its non-specic nature and the probable role of macrophages in the
phenomenon. Developmental and ComparativeI mmunology 9, 419432.
Palmer, R. & Smith, P. R. (1980). Studies on vaccination of Atlantic salmon
against furunculosis. I n Fish Diseases; Third COPRAQ-Session (W. Ahne, ed.)
pp. 107112. Berlin, Heidelberg, New York: Springer-Verlag.
Paterson, W. D. & Fryer, J . L. (1974). I mmune response of juvenile Coho salmon
(Onchorynchus kisutch) to Aeromonas salmonicida cells administered intra-
peritoneally in Freunds complete adjuvant. J ournal of the Fisheries Research
Board of Canada 31, 17511755.
Paterson, W. D., Lall, S. P., Airdrie, D., Greer, P., Greenham, G. & Poy, M. (1985).
Prevention of disease in salmonids by vaccination and dietary modication.
Fish Pathology 20, 427434.
Phipps, B. M., Trust, T. J ., I shiguro, E. E. & Kay, W. W. (1983). Purication and
characterization of the cell surface virulent A-protein from Aeromonas
salmonicida. Biochemistry 22, 29342939.
Press, C. McL., Reitan, L. J . & Landsverk, T. (1995). Antigen retention and
enzyme reactivity in the spleen of Atlantic salmon, Salmo salar L., following
administration of injectablefurunculosis vaccines. J ournal of Fish Diseases 18,
199210.
Ramon, G. (1926). Procds pour accroitre la production des antitoxines. Annales de
lI nstitut Pasteur 40, 110.
Rrstad, G., Aasjord, P. M. & Robertsen, B. (1993). Adjuvant e#ect of a yeast glucan in
vaccines against furunculosis in Atlantic salmon (Salmo salar L.). Fish &
Shellsh I mmunology 3, 179190.
Strmsheim, A. (1993). Genetic variation in specic and non-specic immune traits in
Atlantic salmon. Thesis for the degree of Doctor scientiarum, Norwegian
College of Veterinary Medicine, 1993. 125pp.
Thuvander, A., Fossum, C. & Lorenzen, N. (1990). Monoclonal antibodies to salmonid
immunoglobulins: characterization and application to immunoassays.
Developmental and ComparativeI mmunology 14, 415423.
Trust, T. J . (1986). Pathogenesis of infectious diseases in sh. Annual Review of
Microbiology 40, 479502.
Udey, L. R. & Fryer, J . L. (1978). I mmunization of sh with bacterins of Aeromonas
salmonicida. MarineFisheries Review 40, 1217.
Vanselow, B. A. (1987). Theapplication of adjuvants toveterinary medicine. Veterinary
Bulletin 57, 881896.
350
P. J . MI DTLYNG ET AL.