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INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, OCt. 1986, p. 518-520
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Copyright 0 1986, International Union of Microbiological Societies
Vol. 36, No. 4
Vibrio salmonicida s p . nov., a New Fish Pathogen
E. EGIDIUS,' R. WIIK, K. ANDERSEN, K. A. HOFF, AND B. HJELTNES
Institute of Marine Research, 5000 Bergen, Norway
A disease persisting in Norwegian salmonid cultures since the late 1970s is caused by a Vibrio-like bacterium.
This bacterium belongs to the genus Vibrio, but it differs from all previously described species of this genus. The
name Vibrio salmonicidu sp. nov. is proposed. The type strain is strain NCMB 2262 (= HI 7751).
In 1979 a new disease appeared in Norwegian salmonid
farms around the island of Hitra south of Trondheim. This
disease persisted at Hitra and northward until the late
autumn of 1983, when it struck southward to Stavanger. In
particular, the Bergen region, which has the highest density
of fish farms, was badly hit.
The disease is characterized by anemia and extended
hemorrhages, especially in the integument surrounding the
internal organs of the fish. There is a general septicemia,
often with large numbers of bacteria in the blood of moribund and newly dead fish. Isolations were attempted from
the blood and kidneys.
This disease occurs mainly in late autumn, winter, and
early spring and has been called cold-water vibriosis or Hitra
disease (7). The disease has also been described under the
term hemorrhagic syndrome (13) with undefined etiology.
We isolated a Vibrio-like bacterium from all of the fish which
we examined. Tests with these isolates fullfil Koch's postulates and indicate the bacterial etiology of the disease (7).
Characterization of the bacterium strongly indicated that it
is a new species of Vibrio.
MATERIALS AND METHODS
Bacterial strains and growth. Unless otherwise stated,
substrates were prepared with 1.5% NaCl, and incubation
was at 15°C. The general substrates used throughout this
work were nutrient agar (Oxoid Ltd., London, United Kingdom) supplemented with 5% human blood (NBA), and
tryptone soy broth (TSB) (Oxoid) supplemented with NaCl
to a final concentration of 1.5%.
Similar bacteria were isolated from diseased fish all along
the Norwegian west coast. Strain HI 7751, which was isolated
from a farmed Atlantic salmon in the Bergen region in late
1983, was chosen as the type strain and is described here.
Cultures were isolated from moribund or newly dead fish
on NBA which was incubated at 15°C. Colonies were
detected after 1to 5 days.
Salinity optimum and range. The optimum salinity was
determined in TSB, which basically contained 0.5% NaCl.
NaCl was added to TSB to give final concentrations of 1.0,
1.5, 2.0, 2.5, 3.0, 3.5, and 4.0%. Salt-free TSB was made
from the standard ingredients by omitting NaCl. Growth was
measured nephelometrically. To test the survival of the
bacteria in the presence of different NaCl concentrations,
the cultures were streaked onto NBA after 11days, and the
plates were observed for 2 weeks. To test for an NaCl
requirement, modified TSB was made with 1.5% KCl; TSB
containing 1.5% NaCl was used as a control.
Temperature optimum and range. The temperature optimum was determined on an NBA plate (50 by 10 cm) on
* Corresponding author.
which a temperature gradient was set up by using a thermostatic cooler at one side and a heater at the other. The growth
limits were determined by inoculation onto NBA plates
which were incubated at different temperatures.
Sensitivity. Sensitivity disks were made by transferring 10
pl of a saturated solution of 2,4-diamino-6,7-di-isopropylpteridine phosphate (vibriostatic agent 01129) in water to a
sterile disk, which was then placed in the center of an NBA
plate covered with bacteria.
Sterile antibiotic sensitivity disks (AB BIODISK; Solna,
Sweden) were used to determine novobiocin sensitivity (5
pg/disk).
Biochemical tests. Biochemical activity was tested by using
the API 50CH system (API System S.A., La balme les
Grottes, France) with sterile NaCl added to the fluid to a
concentration of 1.5%. Strains were also tested by using the
API 20B system supplemented with sterile NaCl at a final
concentration of 2.0%. The presence of cytochrome oxidase
was tested in 24-h NBA cultures, which were transferred to
filter paper with 1%n,n-dimethyl-p-phenylendiaminehydrochloride (12). The reduction of nitrate was tested by using
the method of Conn (5). Indole was tested in TSB after 48 h
with Kovacs reagent (5). Fermentative metabolism and
oxidative metabolism were tested in Hugh-Leifson medium
supplemented with 1% glucose by using bromthymol blue as
an indicator (4); tests were read after 10 days. Chitinase
production was tested on chitin agar plates containing minerals and 0.4% dried, purified chitin by using the method of
Hsu and Lockwood (11).Production of lipase was tested by
the method of Harrigan and McCance (9), and the ability to
decarboxylate arginine was tested on Mgller decarboxylase
medium (4).
Serology. Rabbit sera were produced against some of the
bacterial strains (see Fig. 3). An enzyme-linked immunosorbent assay was performed by the method of Bratthall et al.
(2), with some modifications. A 96-well microtitration plate
(InterMed; Nunc, Roskilde, Denmark) was coated with
bacterial cells. Rabbit sera were diluted 1:4,000 in phosphate-buffered saline (pH 7.2) containing 0.05% Tween 20
and 0.5% bovine serum albumin (Sigma Chemical Co., St.
Louis, Mo.) . Goat anti-rabbit immunoglobulin G-horseradish peroxidase conjugate (Bio-Rad Laboratories, Richmond, Calif.) was diluted 1:3,000 in phosphate-buffered
saline containing Tween 20 and bovine serum albumin. A
100-p1 portion of a solution of 40 mg of 0-diaminobenzene
dihydrocloride [E. Merck AG, Darmstadt, Federal Republic
of Germany] in 100 ml of phosphate-citrate buffer [pH 5.01
with 40 pl of water added immediately before use was added
to each well. The reaction was stopped by adding 50 pl of 2.5
NHZS04 to each well after 3 min, and the optical density at
492 nm was read with a model MR 600 microplate reader
(Dynatech Laboratories, Inc., Alexandria, Va.).
518
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VIBRIO SALMONICIDA SP. NOV.
VOL.36, 1986
FIG. 1. V. sulrnonicidu cell prepared for scanning electron microscopy by uranyl acetate staining , showing a polar tuft of nine
sheathed flagella. Prepared by T. Rolfsen, University of Bergen,
Bergen, Norway. ~ 7 , 1 2 0 Bar
.
= 1 Fm.
Pathogenicity tests. Tests for pathogenicity by intraperitoneal injection were performed by using the method of
Egidius et al. (7). Tests for waterborne infection were
performed as described by Egidius and Andersen (6) at 8 to
9°C.
RESULTS AND DISCUSSION
Strain HI 7751T (T = type strain) is a gram-negative,
facultatively anaerobic, curved, motile rod-shaped organism. It is oxidase positive and strongly susceptible to
vibriostatic agent 0/129; Na+ is required for growth, and the
guanine-plus-cytosine content is 42 mol% (14). Growth is
oxidative or fermentative. These characteristics, together
with its previously described morphology and its biochemical reactions, place this organism in the genus Vibrio
(Pacini). The characteristics of this organism differ from
those of previously described Vibrio species. We propose
the new species described below.
Vibrio salmonicida sp. nov. Vibrio salmonicida (sal. mon. i.
ci' da. N.L.n. salmon salmon; L.v. caedo to kill; N.L. adj.
salmonicida salmon killer). During initial isolation growth on
artificial substrates may take 3 to 5 days. After a few
passages on such substrates, good growth is obtained within
48 h. In many cases apparently pure cultures are obtained
directly from the kidneys of moribund or newly dead fish.
Cells from 24-h cultures on NBA are curved rods which
are 0.5 by 2 to 3 pm. When they are isolated, strains may
show a high degree of pleomorphism. Electron micrographs
of cells from 24-h tryptone soy agar plates show the presence
of at least nine polar sheathed flagella (Fig. 1). No lateral
flagella are observed.
Colonies on NBA are small, smooth with entire edges, and
grayish; neither pigmentation nor swarming is observed. No
hemolysis of either human or sheep blood.
The optimum salinity for growth is 1.5% NaCl. Growth
occurs at NaCl concentrations between 0.5 and 4.0% (Fig.
2). After 11 days growth is obtained only from cultures
containing 3.0, 3.5, and 4.0% NaC1, which may indicate a
higher survival capacity at higher salinities. Na' is essential
for growth.
Growth occurs between 1 and 22°C; optimum growth
519
occurs at 15°C. A slight increase in maximal temperature can
occur after many passages on an artificial substrate.
Cultures are sensitive to vibriostatic agent 0/129, with
zones of inhibition up to 30 mm in diameter, and are
relatively resistant to novobiocin (diameter of inhibition
zone, 18 mm).
Biochemical reactions. Catalase and cytochrome oxidase
are present. Citrate cannot be used as the only carbon
source; nitrates are not reduced. Acetoin, arginine, H*S, and
indole are not produced. Chitinase, P-galactosidase, gelatinase, lipase, and urease are not present. D-Fructose, Dgalactose, D-glucose, maltose, ribose, and trehalose are
utilized, as are N-acetylglucosamine, gluconate, and mannitol. Glycerol is utilized slowly. The following compounds
are not utilized: D-arabinose, L-arabinose, D-cellobiose, Dfucose, L-fucose, f3-gentibiose, lactose, D-lycose, D-mannose,
melezitose, melibiose, D-raffinose, rhamnose, sucrose, Lsorbose, D-tagatose, D-turanose, D-xylose, L-xylose,
adonitol, D-arabitol, L-arabitol, amygdalin, arbutin, dulcitol,
erythritol, esculin, a-methyl-D-glucoside, 2-keto-gluconate,
5-keto-gluconate, glycogen, inositol, inulin, a-methyl mannoside, salicin, sorbitol, starch, xylitol, and p-methyl
xyloside.
The reactions of the type strain have been tested by using
API systems repeatedly during the 2 years since its isolation
and seem to be stable, even though the reactions tend to
become weaker. Loss of the ability to use gluconate is the
only exception.
Seven other isolates from diseased fish from different
localities all along the Norwegian west coast and one isolate
from Shetland (3) have also been tested by using the API
systems. The results are similar to those obtained with the
type strain, with a few exceptions. Only four of the strains,
including the one from Shetland, can utilize galactose. The
Shetland strain can utilize D-mannose, and its utilization of
glucose varies. Two of the strains do not utilize mannitol.
The strains vary in their ability to utilize ribose, and the
reaction usually is weak. Holm et al. (10) found the same
biochemical pattern as described above for the type strain,
with the exception of slow utilization of cellobiose and
utilization of gentiobiose and D-mannose.
Serology. No cross-reaction is observed with the enzymelinked immunosorbent assay between V. salmonicida and
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0.5
1
1.5
2
2.5
3
3.5
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SALINITY 0
FIG. 2. Growth of V. salmonicidu NCMB 2262= in TSB at
different salinities. Symbols: 0, after 24 h; +, after 59 h.
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520
INT. J. SYST.BACTERIOL.
EGIDIUS ET AL.
outbreaks occur at lower water temperatures, which is in
agreement with the temperature ranges which we found for
growth. Disease outbreaks southward along the Norwegian
Coast in 1983 might be related to generally lower water
temperatures in this region during that year.
V. salmonicida (type strain HI 7751) has been deposited
with the National Collection of Marine Bacteria, Aberdeen,
Scotland, as strain NCMB 2262.
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NCMB 2129 NCMB 2133 NCMB 6
NCME
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HI 7953
BACTERIAL STRAINS
FIG. 3. Serological differences between V. anguillarum and V .
salmonicida. Each group of bars represents the immunological
(enzyme-linked immunosorbent assay) reactions between selected
bacterial strains and rabbit antisera produced against strains NCMB
2129, NCMB 2133, NCMB ST, NCMB 2262, and HI 7953. The
reactions were measured in absorbance units by using the enzymelinked immunosorbent assay technique. Strains NCMB 2129,
NCMB 2133, and NCMB 6Tare V. anguillarum strains, and strains
NCMB 2262 and HI 7953 are V. salmonicida strains.
three strains of Vibrio anguillarum (Fig. 3 ) . V . salmonicida
is serologically distinct from V . anguillarum.
Pathogenicity. Intraperitoneal injection of broth cultures of
V . salmonicida into both rainbow trout and Atlantic salmon
and challenge through water reproduce symptoms identical
to those of natural outbreaks. In each instance the bacterium
can be reisolated. Atlantic salmon appear to be more susceptible than rainbow trout to the disease.
Of 22 previously described species, only Vibrio fisheri (l),
Vibrio logei (l),Vibrio marinus (l), and Vibrio damsela (8)
have guanine-plus-cytosine contents that are significantly
lower than 44 mol%, as is the case with V. salmonicida (14).
These four species also are the only species which have the
same temperature pattern for growth as V . salmonicida.
Pathogenicity may be lost by passage on artificial substrates but can be regained by passage through fish. Disease
LITERATURE CITED
1. Baumann, P., and R. H. W. Schubert. 1984. Family 11.
Vibrionaceae Vernon, p. 516-538. In N. R. Krieg and J. G. Holt
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2. Bratthall, D., L. Gahnberg, and B. Krasse. 1978. Method for
detecting LGA antibodies to Streptococcus mutans serotypes in
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3. Bruno, D. W., T. S. Hasting, A. E. Ellis, and R. Wotten. 1985.
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Cold-water vibriosis or Hitra-disease in Norwegian salmonid
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11. Hsu, S. C., and J. L. L. Lockwood. 1975. Powered chitin agar as
a selective medium for enumeration of Actinomycetes in water
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(haemorrhagic syndrome) in Norwegian salmon farming: present status, p. 223-229. In A. E. Ellis (ed.), Fish and shellfish
pathology. Academic Press, Inc., New York.
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salmonicida sp. nov. to other fish-pathogenic vibrios. Int. J.
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