AN ABSTRACT OF TEE ThESIS OF
Somsak
PipoDtinvo
for the degree of
Presented on
Master of Science
Sentember 15.. 1987
in
Fisheries
Title:
BIOCHEMICAL AN]) SEROLOGICAL COMPARISON OF SELECTED VIBRIO
SPP. ISOLATED FROM FISH
Abstract approved:
Redacted for Privacy
I
Dr.
James R. Winton
Nine isolates of bacteria recovered from fish dying at
marine facilities were collected from different geographic areas.
The strains included: an isolate from chinook salmon (Oncorhvnchus tsbavvtscha) reared in net pens in New Zealand, an isolate
from chum salmon (Oncorhvnchus keta) held at a laboratory in
Oregon, USA., and seven strains recovered from tilapia (Oreochro-
sDilurus), silvery black porgy (Acanthoagrus cuvieri), and
greasy grouper (Eninenhelus tauvina) cultured in Kuwait.
All
isolates were characterized by examination of morphological and
biochemical properties and were confirmed to be members of the
genus Vibrio.
All, isolates differed phenotypically from each other, from
vibrios known to be pathogenic for fish, and from other named
Vibrio species.
Analysis of key phenotypic characteristics used
to establish existing species suggested that the isolates tested
were new Vibrio species.
Four of the isolates (two from coldwater fish and two from
warmwater fish) were selected for further study.
This included
determination of percent guanine plus cytosine (%G+C), comparison
of growth characteristics, analysis of major 0 antigens and
testing of pathogenicity.
The four isolates examined had an absolute requirement for
NaCl.
Optimum growth temperatures varied among the isolates and
were consistent with the temperature optima of the hosts from
which the isolates were obtained.
Serological analysis using slide agglutination, microtiter
agglutination, and Ouchterlony double diffusion tests detected
specific thermostable (0) antigens unique for each of the four
isolates.
A coon minor antigen was observed between two of the
other isolates from Kuwait.
Experimental infections were produced in fingerling rainbow
trout (Salmo gairdnerfl using intraperitoneal injection of the
four isolates.
The pathogenicity of the two isolates from Kuwait
The strains
was higher than that of the two salmonid isolates.
fromKuwaitwere used to challenge juvenile chinook salmon by
vaterborne exposure.
The pathology produced by infection was
characteristic Cram-negative hemorrhagic septicemia.
Biochemical and Serological Comparison of
p. Isolated from Fish
Selected Vibrio
by
Somsak Pipoppinyo
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed September 15, 1987
Commencement June 1988
APPROVED:
Redacted for Privacy
Assistant Professor of Fisheries in charge of major
Redacted for Privacy
Read of Department 'of Fisheries and Wildlife
Redacted for Privacy
Dean of GraduatI Echool
Date thesis is presented
Typed by Somsak Pipoppinyo for
Seotember 15. 1987
Somsak Pi0000invo
ACKNOWLEDGEMENTS
I would like to express my deep appreciation to the many
people who have helped to make this work possible:
To Dr. J. L. Fryer and
Dr. J. R. Winton for the opportuni-
ty to work in their laboratory; and especially to Dr. Winton, my
major professor, for his tremendous assistance, guidance, patience, and encouragement in the face of imminent deadlines.
To the Royal Thai Government and the Maejo Institute of
Agricultural Technology, Thailand, for financial support throughout this study.
To Dr. J. S. Rohovec for many helpful technical suggestions
and editorial comments in the preparation of this manuscript.
To Dr. R. E. Olson and Dr. L. E. Lampila for their help in
the final preparation of this thesis.
To Craig Banner for assistance with the percent guanine plus
cytosine determinations.
Special thanks to Cathy Lannan, Kellee Roberti, Yuanan Lu,
and Jerri Hoffmaster for their friendship, assistance, and constant encouragement throughout this study; and to the other members of the fish disease group: Tony Ainandi, Cindy Arakawa,
Warren Groberg, Rich }Iolt, John Kaufman, Scott LaPatra, and Dan
Rockey whose help and friendship will always be remembered.
Finally, I wish to express my deep gratitude to my parents
for their love, understanding, and constant encouragement.
This work is a result of research sponsored by Oregon Sea
Grant through NOAA Office of Sea Grant, Department of Commerce,
under Grant No. 1985-86 (FY86) NA85AA-D-SG095.
TABLE OF CONTENTS
Page
INTRODUCTION
i
LITERATURE REVIEW
3
MATERIALS AND METHODS
18
Bacterial Strains, Culture Media, and Maintenance
18
Phenotypic Characteristics
20
Morphology, Motility, and Gram Stain Reaction
Biochemical Tests by API-20E Strips
Biochemical Tests by Conventional Methods
Growth Characteristics
Growth Curves and Mean Generation Time
Growth at Different Temperatures
Growth at Different Concentrations of Sodium
Chloride
Serological Tests
Production of Rabbit Antiserum
Slide Agglutination Tests
Determination of Agglutinating Antibody Titer
Ouchterlony Double Diffusion Tests
Percent Guanine Plus Cytosine Determination
Isolation of DNA
Determination of Percent Guanine Plus Cytosine
Virulence and Pathogenicity Tests
Injection Challenge
Waterborne Challenge
20
21
21
23
23
23
24
24
24
25
26
27
28
28
29
30
30
31
RESULTS
32
Phenotypic Characteristics
32
Growth Characteristics
Growth Curves and Mean Generation Times
Growth at Different Temperatures
Growth at Different Concentrations of Sodium
Chloride
Serological Tests
Slide Agglutination Tests
Determination of Agglutinating Antibody Titers
Ouchterlony Double Diffusion Tests
32
32
35
35
43
43
43
46
Percent Guanine Plus Cytosine Determination
46
Virulence and Pathogenicity Tests
50
Injection Challenge and Fifty Percent Lethal
Dose (LD50)
Waterborne Challenge
50
56
DISCUSSION
63
SUMMARY AND CONCLUSIONS
71
BIBLIOGRAPHY
73
LIST OF FIGURES
Page
Figure
Growth curve of NZ isolate in tryptic soy broth
at 18°C plotting viable cells/mi versus optical
density.
36
2
Growth curve of CHIJ isolate in tryptic soy broth
at 18°C plotting viable cells/mi versus optical
density.
36
3
Growth curve of Ku-2 isolate in tryptic soy broth
at 18°C plotting viable cells/nil versus optical
density.
37
4
Growth curve of Ku-4 isolate in tryptic soy broth
at 18°C plotting viable cells/nil versus optical
density at.
37
5
Growth of the 4Z isolate in tryptic soy broth
incubated at different temperatures.
38
6
Growth of the C}IU isolate in tryptic soy broth
incubated at different temperatures.
38
Growth of the Ku-2 isolate in tryptic soy broth
incubated at different temperatures.
39
8
Growth of the Ku-4 isolate in tryptic soy broth
incubated at different temperatures.
39
9
Growth of Vibrio anguillarum in tryptic soy
broth incubated at different temperatures.
40
10
Growth of the NZ isolate in tryptone glucose
yeast broth at different concentrations of NaCl
incubated at 18°C.
40
11
Growth of the CHU isolate in tryptone glucose
yeast broth at different concentrations of NaCl
incubated at 18°C.
41
12
Growth of the Ku-2 isolate in tryptone glucose
yeast broth at different concentrations of NaCl
incubated at 18°C.
41
13
Growth of the Ku-4 isolate in tryptone glucose
yeast broth at different concentrations of NaC1
42
1
7
incubated at 18°C.
14
Growth of Vibrio anguillarum in tryptone glucose
yeast broth at different concentrations of NaC1
incubated at 18°C.
42
15
Ouchterlony double diffusion plates. Antigens
in the outer wells were reacted with antisera
in the center wells.
47
16
Characteristic hemorrhagic septicemia in rainbow
trout injected by isolates studied.
57
17
External pathological signs in chinook salmon
exposed to Ku-2 isolate.
61
LIST OF TABLES
Page
Table
1
Characteristics of members of the genus Vibrio
that have been reported to be pathogenic for
5
fish.
2
Source of Vibrio isolates used in this study.
19
3
Phenotypic characteristics of Vibrio isolates
used in this study compared with Vibrio an2uillarum and V. ordalii.
33
4
Results of slide agglutination tests using rabbit
antisera against Vibrio anuillarum,
ordalii,
NZ, CILU, Ku-2, and Ku-4 reacted with homologous
and heterologous antigens.
44
5
Agglutinating antibody titers of rabbit antisera
ordalii, NZ, CHU,
against Vibrio anguillrum,
.
Ku-2, and Ku-4 when reacted with the homologous
and heterologous antigens.
45
6
Percent guanine plus cytosine (%G+C) values of
the NZ, CHU, Ku-2, and Ku-4 isolates using
cherichia coli (WP2) as the reference strain.
51
7
Mortality and mean day to death of rainbow trout
injected with different numbers of NZ cells.
52
8
Mortality and mean day to death of rainbow trout
injected with different numbers of CHU cells.
53
9
Mortality and mean day to death of rainbow trout
injected with different numbers of Ku-2 cells.
54
10
Mortality and mean day to death of rainbow trout
injected with different numbers of Ku-4 cells.
55
11
Mortality and mean day to death of chinook salmon waterborne challenged with Ku-2.
59
12
Mortality and mean day to death of chinook salmon waterborne challenged with Ku-4.
60
BIOCHEMICAL AND SEROLOGICAL COMPARISON OF
. ISOLATED FROM FISH
SELECTED VIBRIO
INTRODUCTION
Members of the genus Vibrio are wide-spread in nature,
occuring principally in marine and estuarine environments
These bacteria have been
(Baumann et al., 1984; Colvell, 1984).
isolated from diseased fish of various species throughout the
world and are a leading cause of mortality among cultured marine
fishes (Anderson and Conroy, 1970; Austin and Austin, 1987).
The
genus Vibrio is formed from a large complex of strains (Baumann
et al., 1984).
Of the twenty named species, nine (!. alginoly-
ticus, !. anguillarum,
ordalii,
s
.
carcharae,
parahaemolvticus,
.
cholerae,
. damsela,
.
. salmonicida and !. vulnificus)
have been described as pathogens of fish (Coiwell and Grimes,
1984; Egidius et al., 1986; Austin and Austin, 1987).
against
.
Vaccines
anguillarum and I. ordalii are commercially available
and are effective in reducing mortality caused by these species.
In addition to the named vibrio species, several workers have
reported the isolation of fish pathogens which may represent new
species (McCarthy, 1976; Strout et al, 1978; Tajima et al.,
1986a, 1986b).
This study used nine vibrio isolates obtained
from fish dying at warmwater and coldwater marine facilities in
different geographic areas (New Zealand, United States, Kuwait)
to determine the relatedness of the strains found in diverse
types of mariculture.
It was the purpose of this study to: (1) compare the
morphological, biochemical, physiological and serological characteristics of the isolates with each other and with reference
strains of V. anguillarum and V. ordalii, (2) determine the
taxonomic placement of the isolates within the genus Vibrio, (3)
evaluate the pathogenic potential of selected isolates, and (4)
examine the characteristics of the strains to determine if
isolates from a given geographic area or type of fish shared
similar traits.
Morphological examination and selected biochemical and phyiological tests were performed to confirm the isolates were members of the genus Vibrio.
Those characteristics which are im-
portant for separating isolates into named species (Baumann et
al., 1984) were given special attention.
These tests confirmed
all isolates should be included within the genus; however, the
isolates were different from the 20 established species.
Sero-
logical tests were used to determine antigenic relationships
among the isolates.
These tests showed that the strains were not
clearly related to each other nor to
ordalii.
anguillarum or y.
Selected isolates were tested for pathogenicity in
fingerling rainbow trout (Salmo gairdneri) and juvenile chinook
salmon (Oncorhynchus tshawvtscha) by injection and waterborne
exposure.
These studies showed variations in virulence between
the isolates and suggested that additional factors such as temperature, salinity, stress or presence of other pathogens may be
important factors in pathogenesis.
3
LITERATURE REVIEW
The Genus Vibrip
The genus Vibrio, as described by Baumann et al. (1984) and
Coiwell and Grimes (1984), is composed of Grain-negative, straight
to curved rods, 0.5-0.8 urn in width and 1.4-2.6 urn in length.
liquid media, Vibrio
In
. are motile by monotrichous or multitri-
chous polar flagella which are enclosed in a sheath continuous
with the outer membrane of the cell wall.
On solid media, some
species possess nonsheathed lateral flagella which play a role in
attachment to surfaces and appear to be involved in swarming.
Biochemical features of the genus include fermentation of
glucose, usually without the production of gas; sensitivity to
the vibriostatic compound, 0/129 (2,4-diamino 6,7-diisopropylpteridine); ability to utilize D-glucose, D-fructose, maltose and
glycerol; and an absolute requirement for sodium chloride.
species are oxidase positive.
30°C.
Most
All grow at 20°C and some grow at
The molZ G+C of the DNA ranges from 38-51%.
. are aquatic areas with a
The natural habitats of Vibrio
wide range of salinities which are common in marine and esturine
environments, and on the surfaces and in the intestinal contents
of marine animals.
Several species are pathogenic f or man as
well as for marine vertebrates and invertebrates.
The genus Vibrip, as it is currently recognized, contains
over 20 species (Baumaun et al., 1984).
.
alginolyticus,
damsela,
anguillarum,
Nine species of Vibrio,
. carchariae,
. cholerae,
. ordalii, !. parahaemolvticus, !. salmonicida, and
ri
vulnificus (Table 1) have been described as pathogens of fish
(Colwell and Grimes, 1984; Egidius et al., 1986; Austin and
Austin, 1987).
Vibrio alginolyticus
Vibrio alginolvticus has been reported to cause mortalities
following handling of farmed seabream (Sparus aurata) in Israel.
However, fish could not be infected under experimental conditions
(Colorni et al., 1981).
The organism has also been reported to
cause mortalities in young red seabream (Pagrus major) (Iwata et
al., 1978) and has been associated with ulcer disease (Akazawa,
1968).
Furthermore, L. alginolyticus has been reported as a
secondary invader associated with "red spot", a disease caused by
y. anguillarum in sea mullet (Mugil cephalus) (Burke and Rodgers,
1981).
Gross pathology was described as a typical septicemia by
Colorni et al. (1981).
Infected fish became inactive and dark,
lost scales, and developed ulcers.
The liver, swim bladder,
peritoneum, and capillaries in the intestine walls were congested.
Simultaneously, the intestine and gall bladder were
distended with clear fluid and bile, respectively.
Anemia and
gill erosion were also observed.
Some characteristics that distinguish 'L alginolyticus from
other species of Vibrio include: swarming on solid complex media,
growth at 40°C, tolerance to 10% NaC1, positive Voges-Proskauer
reaction, and utilization of sucrose, valerate, L-leucine and
L-tyrosine.
Vibrio alginolyticus does not produce arginine
5
Table 1.
Characteristics of members of the genus Vibrio that
have been reported to be pathogenic for fish.
.1
C"1
0
0
Ca
-i
o
0
U)
H
0
H
U)
Cl)
l))
rI
rl
>'
H
Ci
,-4
a)
4J
4
H
o
H
H
H
H
Ct
H
C
Ct
Ci
4
r-4
CT)
CT)
CT)
Ci
(1)
H
a)
Motility
+
+
Gramstain
-
+
+
+
+
+
+
0
Voges-Proskauer reaction
Gelatinase
Utilization of:
Glucose
Mannitol
Inositol
Sorbitol
Rhamnose
Sucrose
Melibio8e
Amygdalin
Arabinose
Cellobiose
D-Gluconate
Gas from D-glucose
ReductionofN0toN0
Lipase
Cytochrome Oxidase
Sensitivity to 0/129
Pigmentation
Swarming on solid media
tt
,.Ct
Ci
.I
Ci
Ct
.Ct
Ct
Ct
CT)
0
+
-
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
n
n
n
n
-
+
+
+
+
+
+
+
+
+
+
+
-
Ci
Ci
Ci
H
H
H
H
4-1
0
H
4-1
H
CT)
U)
I
+
I
I
+
+
+
t
+
-
-
-
-I
+
+
+
+
+
+
+
+
+
Ct
.
l
+
+
H2Sproduction
Indoleformation
CC
H
r
H
.
j
Urease
Tryptophane deaminase
Ct
-4
Characteristics
PHB accumulation
-D-galactosidase (ONPG)
Arginine dihydrolase
Lysine decarboxylase
Ornithine decarboxylase
Citrate utilization
Ha)
Ct
c
0
r4
H
Ci
+
fl
n
ii
fl
+
+
+
+
+
+
-
d
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
-
d
+
+
-
v
-
n
+
-
+
+
+
+
+
+
+
+
+
d
-
+
-
-
-
-
-
+
-
-
+
+
n
n
+
-
+
V
+
-
+
+
-
+
-
fl
+
+
+
+
-
-
V
+
+
-
+
+
+
+
+
+
-
V
+
n
+
+
-
+
+
+
+
+
+
+
+
n
+
+
+
+
n
+
+
+
+
+
-
-
+
05
(1
'.
-0 P1
P1
0 0
0 0
'.0
0)
P1P1
CD
00)0)
0305
0
rP
r
CD
I-..
0)
mm
P-.P-..
rPrP
03.
CDDQ
0)
CD
CD0
I-. CD
-. P...
.0
CD03P1P1
rP
0
-. P1 P
030)0)
P1 ras rrP
03
Pti P-h
rl'.4
pa p...
I- 03
0 P-
CD
P1 0P1 00
0)rP
rrr03 (D(D
P1P1
I1O 0
'.
I
I-.. 0 B B
005
as
I-s
r1
05P1
P1
CD
flflflhIH
++I
I
I+++I
111+1
I++I
111+1
V. vulnificus biogroup 1
+++I
V. vulnificus biogroup 2
V. salmonicida
II
I
V. parahaemolyticus
I
+++I
I
V. ordalii
I
I++I
V. cholerae
V. carchariae
V. anguillaruni
V. alginolyticus
03
C)
P...
rP
0)
V. damsela
+++I
CD
P1
I-..
5
C)..
CD
I-..
r1
0
C)
'-S
CD
03
(5
rP
P...
P1
03
C)
I+++I
I I+
I I+I
I
I-+-
C) C) C)
+++I
i-. P-i I_.
C)
4-(3.a
QjtO.P0 0 0 003
++++I
I-
C) C) C) C) C)
0303030303
O0303O
I-.
0
P1
o
7
dihydrolase and can not utilize cellobiose,
-hydroxybutyrate and
Y-aminobutyrate.
Vibrio anguillarum
Vibrio anguillarum, a causative agent of vibriosis, is
considered to be one of the most serious infectious diseases
affecting various kinds of wild and cultured fish throughout the
world (Cisar and Fryer, 1969; Anderson and Conroy, 1970).
The disease is particularly devastating to cultured salt water
However, there have been reports of I. anuillarum
fish.
isolations from fish living exclusively in fresh water, notably
in Japan and Italy (Muroga, 1975; Ohnishi and Muroga, 1976;
Giorgetti and Ceschia, 1982).
The disease caused by I. anguillarum is normally a
generalized septicemia with clinical signs ranging from acute
(mortalities without gross lesions) to subacute (hemorrhaging of
the eyes, gill, vent, skin, and internal organs, blood-tinged
fluid in the body cavity) and chronic (hemorrhagic ulceration of
the skin and underlying muscle) (Bullock et al.,
1971; Amos,
1985).
Vibrio anuillarum was the first recognized pathogen of
fish and was isolated and described by Canestrini in 1893 and
Bergman in 1909 (Rucker, 1959; Groberg, 1981).
There have been
many changes in the nomenclature of this bacterium since its
first isolation.
It has been called Bacterium anguillarum,
Vibrio anguillarum, Vibrio piscium, Achromobacter ichthvodermis,
Pseudomonas ichthvoderinis, and Vibrio ichthyodermis (Post, 1983).
[]
Hendrie et al. (1971) proposed combining all of these strains as
a single species, and the name Vibrio anguillarum was officially
recognized in 1974 (Cowan et al., 1975).
Strains of
.
anguillarum have been studied biochemically
(Nybelin, 1935; Smith, 1961; Hastein and Smith, 1977; Baumann et
al., 1978; Ezura et al., 1980; Kaper et al., 1983; West et al.,
1983), serologically (Pacha and Kiehn, 1969; Harrell et al.,
1976; Strout et al., 1978; Ezura et al., 1980; Kitao et al.,
1983; Tajima et al., 1986a,b; Sorensen and Larsen, 1986), and
genetically (Anderson and Ordal, 1972; Schiewe et al., 1977;
Schiewe and Crosa, 1981).
On the basis of biochemical reactions, Nybelin (1935) (cited
from Austin and Austin, 1987) differentiated Vibrio anguillarum
into two groups:
anguillarum var tvpica (type A) and I.
anguillrum var anguillicida (type B).
Type A formed indole and
produced acid from sucrose and mannitol.
Type B produced neither
acid from these carbohydrates nor indole.
Smith (1961) isolated
a strain of
.
anguillarum which fermented sucrose and mannitol
but did not produce indole.
Using the classification scheme of
Nybelin, he designated the bacterium as a type C strain.
and Smith (1977) isolated
Hastein
anguillarum from Norwegian waters
from a variety species of fish and divided the bacterial isolates
into two groups.
The differentiation of
.
anguillarum into two
groups, based on biochemical reactions, was supported by the work
of Baumann et al. (1978) and Ezura et al. (1980).
Of the 219
isolates classified using numerical taxonomic methods, Kaper et
al. (1983) defined four distinct groups among the isolates iden-
9
tified as V. anguillarum.
This view was reinforced in a later
study by West et al. (1983).
Further work is necessary to clari-
fy taxonomic status surrounding these vibrios pathogenic for
fish.
.
Austin and Austin (1987) have updated the description of
anguillarum in Bacterial Fish Pathoens: Disease j
Farmed
Wild Fish as:
cream colored, round, raised, entire, shiny colonies
comprising short (0.5 x 1.5 urn), fermentative, Gram-
negative rods, which are motile by single polar
-galactosidase
H2S, lysine or
ornithine decarboxylase, phenylalanine deaminase or
urease are produced. Along with most other vibrios,
flagella.
Catalase, oxidase, indole,
and arginine dihydrolase, but not
sensivity is displayed to the vibriostatic agent,
A positive result is usually recorded for the
Voges Proskauer reaction, but not for the methyl red
test. Gelatin, DNA, lipids and starch, but not aescul0/129.
in, are degraded.
Nitrates are reduced.
Growth occurs
at 15-37°C, and in 0.5 to 3% (w/v) but not 0 and 7%
Citrate, malonate and tartrate
(w/v) sodium chloride.
are utilized. The organisms produce acid from
arabinose, cellobiose, galactose, glycerol, maltose,
mannitol, sorbitol, sucrose, and trehalose, but not
from adonitol, dulcitol, erythritol, inositol, lactose,
melibiose, raffinose, rhamnose, salicin or xylose.
Results of serological studies further complicated the
classification of
. anguillarum.
Pacha and Kiehn (1969)
investigated serological relationships among marine vibrios
isolated from herring and various salmonid species in the Pacific
Northwest, and from cod and finnock in Europe.
They suggested
that three serotypes composed of Pacific Northwest vibrios
(serotype 1), European vibrios (serotype 2), and Pacific herring
vibrios (serotype 3) were distinguishable based on analysis of
heat-stable (0) antigens.
Harrell et al. (1976) isolated a
strain of Vibrio which was biochemically and serologically
different from the Pacific Northweast archetype
. anguillarum
10
(strain 775) previously isolated by Evelyn (1971).
The new
isolate was designated as Vibrio anguillarum biotype II and
strain 775 was designated as Vibrio anguillarum biotype I.
Based on analysis of heat-stable (0) antigens, Strout et al.
(1978) reported the existance of three
anguillarum serotypes
isolated from confinement-reared and feral fishes along the
Two of these isolates were antigeni-
Maine-New Hampshire coast.
cally similar to the West coast strains of
. anguillarum
biotype I and II
In Japan, epizootics caused by
anguillarum have occurred
in several species of fish and many strains of Vibrio have been
On the basis of cross-agglutination and
isolated and examined.
cross-adsorption tests with thermostable (0) antigens, the most
important strains were grouped into three serotypes: 3-0-1,
3-0-2, and 3-0-3 (or A, B, and C) (Ezura et al., 1980; Kitao et
al., 1983; Tajima et al., 1986a).
were antigenically similar to
ordalii) and
.
.
The 3-0-1 and 3-0-3 strains
anguillarum biotype II (Vibrio
anguillarum biotype I, respectively.
The number
of serotypes was increased to eight upon further study (Tajima et
al., 1986b).
Sorensen and Larsen (1986), however, recently
reported the presence of ten 0-antigen serotypes, based upon
examination of 495 isolates.
Studies of
.
anguillarum using DNA hybridization techniques
have been conducted primarily to analyze differences between
anguillarum biotype I and
port reclassification of
species,
.
.
.
.
anguillarum biotype II and to sup-
anguillarum biotype II as a new
ordalii (Schiewe et al., 1977; Schiewe and Crosa,
11
1981; Schiewe et al., 1981).
In addition, Anderson and Ordal
(1972) used DNA hybridization techniques to determine the rela-
tionships among marine vibrios isolated from Japan, Europe, and
Some strains of
the United States.
.
alginolvticus and I. para-
haemolyticus were included in their study.
The results showed
that a number of strains previously identified as
.
parahaemo-
lyticup. based on morphological, biochemical, and serological
evidence, should be considered to be strains of
. anguillarum.
Vibrio carchariae
Vibrio carchariae was originally isolated from a dead sandbar (brown) shark (Carcharhinus
lumbeus) which died at the Na-
tional Aquarium in Baltimore, U.S.A. in 1982 (Grimes et al.,
1984a).
Subsequently,
.
carchariae has been isolated from lemon
sharks (Negaprion brevirostrig) and from the trematodes found on
the skin of these sharks (Grimes et al., 1985).
The disease was described by Grimes et al. (1984b) and
Colwell and Grimes (1984) as a "vasculitis".
Infected animals
were lethargic, anorexic, disoriented, and developed necrotic
subdermal cysts.
Internal signs of the disease included encepha-
litis, meningitis, kidney necrosis, and there was liver and
spleen involvement.
Experimental infection by intraperitoneal
(i.p.) injection caused mortality of dogfish (Sualus acanthias)
within 18 hours and induced severe splenic and hepatic diseases
in lemon sharks (NegaDrion brevirostris) (Grimes et al., 1985).
Vibrio carchariae displays the characteristic of swarming
on agar medium and biochemical reactions are similar to
.
12
alginolyticus (Table 1).
Unlike
alginolvticus,
.
carchariae
can not grow at 42°C; other differences include inability to grow
in the presence of 10% NaC1 and a negative Voges-Proskauer test.
In addition,
.
carchariae is urease positive.
Vibrio cholerae
Serovars of
cholerae, other than serovar 01, are
occasionally isolated from fish.
The non 01 L. cholerae strains
do not react with the 0 antiserum originally designated as 01
In 1977, a Vibrio ap. was isolated
(Colvell and Grimes, 1984).
and demonstrated to be a causative agent of an epizootic in a
wild population of ayu in the Amano River of Japan (Muroga et
The isolate was morphologically and biochemically
al., 1979).
very similar to typical
cholerae, but did not agglutinate
with Ogawa or Inaba antisera.
nated as
.
cholerae non 01.
Therefore, the isolate was desigIn addition, Muroga et al. (1979)
demonstrated 86% DNA homology between their isolate and a
reference strain of
cholerae (NH35A3).
The disease caused by
cholerae non-Ol was characterized by petechial hemorrhaging
on the body surface.
Internally, there was congestion of the
organs (Muroga et al., 1979).
Vibrio damsela
Vibrio damsela was first isolated from ulcerative lesions along
the flank of blacksmith (Chronus punctipinis), one of the damsel
fish, off the coast of southern California (Love et al., 1981).
The bacteria was thought by Love et al. (1981) to have a narrow
13
host range.
Grimes et al. (1984a), however, isolated I. damsela,
carchariae, from a dead sandbar shark (Carcharhinus
along with
damsela to be
plumbeus), and subsequently demonstrated
virulent for the spiny dogfish shark (Squalus acanthias).
The
damsela in blacksmith as described by Love
disease caused by
et al. (1981) induced characteristic skin ulcers usually near the
pectoral fin and on the caudal peduncle.
a size of 5-20 nun in diameter.
These ulcers may reach
Histopathological examination of
these lesions indicated a granulomatous ulcerative dermatitis.
Lesions were characterized by muscle lysis and by histiocytes
present in the dermis and skeletal muscles.
Some characteristics that distinguish
. damsela from other
species of Vibrio include: production of acid and gas from
glucose; decarboxylation of arginine; hydrolysis of urea, gelatin, and chitin; and positive methyl red and Voges-Proskauer
reactions.
Vibrio damsela can not utilize mannitol, inositol,
sorbitol, rhamnose, sucrose, melibiose, arabinose, and
cellobiose.
Vibrio ordalii
Vibrio ordalii was recently proposed as a new species to
include all strains previously classified as
biotype II (Schiewe et al., 1981).
tinguishable from
.
.
anguillarum
This organism was dis-
anguillarum based on: negative Voges-
Proskauer reaction; failure to hydrolize 0-nitro-pheny1--Dgalactopyranoside (ONPG) and starch, to utilize citrate, and to
show arginine dicarboxylase and lipase activity; inability to
14
37°C; and failure to ferment cellobiose, glycerol,
and trehalose.
j2. ordalii induces hemorrhagic septiceinia in fish and
epizootics have occurred in Japan and the Pacific Northwest, USA.
Typical signs of the diseases caused by !. anguillarum and !.
ordalii differ only slightly.
Vibrio ordalii preferentially
colonizes skeletal and cardiac muscle, gills, and the gastrointestinal tract.
In contrast,
.
anguillarum, induces dissemi-
nated infections, with highest numbers of the bacterium in blood,
loose connective tissue, kidney, spleen, gills, and the posterior
gastrointestinal tract (Ransom et al., 1984).
In addition, sev-
eral investigators have observed a marked leukopenia in moribund
fish, suggesting the presence of leukocytolytic factor in
dalii (Ransom et al., 1984; Harbell et al, 1979; Schiewe, 1983).
Vibrio parahaemolyticus
Vibrio
parphaemolvticus was first recognized as the
etiological agent of human seafood-borne disease in Japan in the
1950s (Coiwell and Grimes, 1984).
fish disease is obscure.
The role of this organism in
Vibrio parahaemolyticus is frequently
isolated from both healthy and diseased fish throughout the world
(Colwell and Grimes, 1984).
This organism is occasionally
isolated from skin ulcers of fish, but usually in association
with other vibrios (Yasunaga and Yamamoto, 1977).
Kusuda et al.
(1979) have isolated V. parahaemolyticus in conjunction with
other vibrios from red seabream cultured in Japan.
Gilmour
(1977) recovered this species of vibrio from fish-holding tanks.
15
Vibrio salmonicida
In 1979 a new disease appeared in Norwegian salmonid farms
around the island of Hitra.
The disease persisted at Hitra and
northward until the late autumn of 1983, when it was observed
along the southern coast of Norway.
The disease occurs mainly in
late autumn, winter, and early spring, and so it is called
cold-
water vibriosis or Hitra disease (Egidius et al, 1981; Egidius et
al 1986).
The pathology of the disease is characterized by anemia and
hemorrhaging, especially in the integument surrounding the
internal organs.
The disease has been also termed hemorrhagic
syndrome (Poppe et al., 1985).
There is a general septicemia,
often with large numbers of bacteria in the blood of moribund and
newly dead fish.
Characterization (biochemical and serological tests, DNA
hybridization and C-C content) of this strain indicated that it
was a new species of Vibrip.
The species name tsa1monicidal
has been used to designate this organism (Egidius et al, 1986;
Wiik and Egidius, 1986).
Vibrio vulnificus
Vibrio vulnificus, originally referred to as a lactose
positive vibrio, has been recognized as a highly virulent,
opportunistic human pathogen (Oliver et al., 1983; Joseph et al.,
1982).
Tison et al. (1982) proposed a new
vulnificus
subgroup, biogroup 2, which was pathogenic for eels.
the names
Previously,
. anguillarum type B and I. anguillicida have been
16
used to described this organism (Muroga et al., 1976a, b;
Nishibuchi et al., 1979).
The eel disease caused by
.
vulnificus biogroup 2 was
characterized by swollen lesions on the body and tail.
In ad-
vanced stages of the disease, histopathological changes were observed in the spleen, liver, kidney, heart, gills and intestine
(Miyazaki et al., 1977).
Vibrio vulnificus biogroup 2 was reported to possess several
characteristic differences from
example,
.
vulnificus biogroup 1.
For
vulnificus biogroup 2 did not produce indole, orni-
thine decarboxylase, acid from mannitol and sorbitol, and did not
grow at 42°C.
In addition, the two biogroups did not share com-
mon surface antigens (Tison et al., 1982).
Results of DNA hy-
bridization studies showed 90% DNA homology between
. vulnificus
biogroup 1 and biogroup 2 (Tison et al., 1982) confirming the
taxononlic relatedness of two groups.
Other Vibrio spp. isolated from fish
In addition to the nine species described, several vibrio
isolates that do not conform to the identified species have been
recovered from diseased fish.
In an epizootic among wild eels in
England, McCarthy (1976) described a Vibrio sp. as the causative
agent.
Strout et al. (1978) reported the existence of three
distinct antigenic groups of Vibrio isolated from pen cultured
and feral fishes along Maine-New Hampshire coast.
were strains of
.
anguillarum and I. ordalii.
Two of these
The third group
were not found to be related to any known Vibrio species.
Thirty
18
MATERIALS AND METhODS
Bacterial. Strains. Culture Media.
,
Maintenance
Nine isolates of bacteria recovered from diseased fish
cultured in various geographic areas (New Zealand, United States,
Kuwait) were used in this study.
The first isolate was recovered
from an epzootic among cultured chinook salmon, in New Zealand in
1985, and was designated as NZ.
The second organism, designated
as CRU, was isolated from diseased chum salmon (Oncorhvnchus
keta) held in Fish Disease Laboratory, Marine Science Center,
Newport, Oregon in 1986.
Seven Kuwait isolates designated as
Ku-i, Ku-2, Ku-3, Ku-4, Ku-5, Ku-6, and Ku-7 were recovered from
three species of fish cultured in Kuwait and were provided by Dr.
Victoria Rasheed, Kuwait Institute for Scientific Research.
The
Ku-i, Ku-2, Ku-5, and Ku-7 were isolated from tilapia (Oreochro-
silurus); the Ku-3 were recovered from silvery black porgy
(Acpnthpparus cuvieri); and the Ku-4 and Ku-7 were obtained from
greasy grouper (EDineDhelus tauvina).
LS-174 and
Vibrio anyuil].arum, strain
ordalii, strain MSC-275 were included in this study
as reference strains (Table 2).
Brain heart infusion medium (Bill, Difco) supplemented with
1% NaCl or tryptic soy broth (TSB, Difco) supplemented with 1%
NaCl were used for routine culture of all bacterial strains.
The
solid media were prepared by addition of 1-1.5% agar (Bill agar or
TSA).
Unless otherwise stated, cultures were incubated at 18°C.
Working stocks of bacteria were maintained in Cytophaga
19
Table 2.
Source of Vibrio isolates used in this study.
Isolate
Location
Host
NZ
New Zealand
Chinook salmon
1985
CHU
Marine Science Center
Newport, Oregon
Chum salmon
1986
Ku-i
Kuwait
Tilapia
1985
Ku-2
Kuwait
Tilapia
1985
Ku-3
Kuwait
Silvery black porgy
1986
Ku-4
Kuwait
Greasy grouper
1986
Ku-5
Kuwait
Tilapia
1985
Ku-6
Kuwait
Tilapia
1986
Ku-7
Kuwait
Greasy grouper
1986
LS-174
Lint Slough,
Waldport, Oregon
Chinook salmon
1974
MSC-275
Marine Science Center
Coho salmon
1975
Year isolated
20
seawater medium (tryptone 0.05%, beef extract 0.02%, sodium
acetate 0.02%, and yeast extract 0.05%) with 3.5% Rila salts and
0.4% agar (Difco).
The stock cultures were stored at 4°C and
transferred every two months.
Reference stocks of each isolate
were preserved in a lyophilized state by modification of the
technique described by Floodgate and Hayes (1961).
Each isolate
was incubated in BHI broth at 18°C and harvested when the
culture entered stationary phase.
A 25 ml aliquot of broth
culture was centrifuged (2200 xg, 15 minutes) and the bacterial
pellet was washed three times in 0.O1M phosphate buffered saline
(PBS, pH 7.0).
The pellet was resuspended in 3 ml of sterile
lyophilization medium (1 part nutrient broth, 3 parts bovine calf
serum, and 7.5% EW/VI glucose).
Aliquots (0.25 ml) of the cell
suspension were transferred to sterile lyophilization vials and
rapidly frozen by inunersion into a bath of acetone and dry ice.
The vials were then evacuated and sealed using a lyophilizer
(Virtis Research Equipment, Inc.), checked for vacuum, and stored
at -20°C.
Phenotypic Characteristics
Morphology. Motility. and Gram Stain Reaction
Cell morphology and motility were observed using phase
contrast microscopy of wet mounts containing cells from the
exponential phase of broth cultures.
Motility was also confirmed
using motility medium (Lenette et al., 1985; beef extract .3%,
peptone 1%, NaCl 1.5%, agar 0.4%, pH 7.4).
To determine the Gram
21
reaction) smears from broth cultures were prepared on microscope
slides3 air dried, heat fixed then stained with Huckers modification of the Gram stain (Society of American Bacteriologists3
1957).
Biochemical Tests
API-20E Strips
The API-20E system (Analytab Products Inc.) was used to
determine hydrolysis of o-nitro-pheny1--D-galactopyranoside
(ONPG), arginine dihydrolase, lysine and ornithine decarboxylase,
citrate utilization, production of hydrogen sulfide, urease,
tryptophane deaminase, indole and acetoin production, gelatinase,
utilization of glucose, mannitol, inositol, sorbitol, rhamnose,
sucrose, melibiose, amygdalin, (1+) arabinose, and reduction of
nitrate.
Procedures
were performed as recommended except that
the incubation temperature was 18°C and the incubation period was
extended to 48-72 h.
Biochemical Tests
All tests were repeated three times.
Conventional Methods
Additional biochemical tests (lipase test, oxidationfermentation [OF] test, and 2,4-diamino-6,7 di-isopropyl
pteridine [0/1291 sensitivity test) which are not included in the
API-20E system were performed using procedures described in the
Manual j Clinical Microbiology. Fourth Edition (Lennette et
al., 1985) and Cowan & Steels Manual
Identification QL
Medical Bacteria. Second Edition (Cowan, 1974).
All tests were
performed in triplicate at 18°C unless otherwise noted.
known organisms were included as controls.
Selected
22
Accumulation of refactile poly--hydroxybutyrate (PHB)
granules was tested in a basal medium containing 50 mM tris
(hydroxymethyl) aminomethane hydrochloride (Tris-HC1); 190 mM
NH4C1; 0.33 mM K2HPO43H20; and 0.1 mM FeSO4'7H20.
The basal
medium was disolved in deionized distilled water with addition of
17.5 gIL of Rila salts.
The pH was adjusted to 7.5 before the
final addition of 4 gIL of sodium-DL---hydroxybutyrate (Sigma
Chemical Company) as the carbon source and 0.2 gIL of (NH4)2SO4
as the sole source of nitrogen.
When the cultures were in early
stationary phase, wet mounts were examined using phase contrast
microscopy.
Tests for utilization of carbohydrates were conducted in
Andrade medium (Rodina, 1972).
This medium was prepared as a 1%
solution of indicator in peptone water.
The indicator solution
was made by adding 16.4 ml of 1 N NaOH to 100 ml of 0.5% acidic
fuchsin and the mixture was heated in an autoclave for 5 mm
110°C.
at
Peptone water was prepared by dissolving 10 g peptone
(Difco) and 15 g NaC1 in 1 liter distilled water.
adjusted to 7.6.
The pH was
Stock solutions of carbohydrates to be tested
were prepared as a 10% solution in distilled water, filtersterilized by passage through membrane filters (0.45 micron pore
size, Gelman Sciences 'Inc.) and aseptically added to the heat-
sterilized basal medium to a final concentration of 1%.
The
utilization of carbohydrates was indicated by a change in the
color of the medium from a light straw to a rose red color.
The presence of cytochrome oxidase was tested using
cytochrome oxidase test strips (Pathotec Co.).
Selected single
23
colonies from plates containing 24-48 h cultures were used.
Growth Characteristics
Growth Curves and Mean Generation Time
Growth studies were conducted using four of the nine strains
of Vibrio Lp. (NZ, CHU, Ku-2, and Ku-4).
Flasks containing 200
ml TSB were inoculated with 0.2 ml of an overnight broth culture
and incubated at 18°C on an agitator.
Samples were removed from
the flasks immediately after inoculation and at selected time
intervals. Triplicate plate counts of the samples were performed
using the spread plate method (Brock and Brock, 1978).
The
optical density at 525 nm of each sample was measured against a
reference tube of uninoculated TSB using a Bausch and Lomb
Spectronic 20.
Plots of viable cell numbers versus time and cell
numbers versus optical density were constructed for each of the
four strains.
Mean generation times were calculated using the
formula and method described by Stainer et al (1970).
Growth at Different Temperatures
To determine the effect of temperature on the growth of four
selected isolates (NZ, CHU, Ku-2, and Ku-4), replicate tubes containing 5 ml TSB were inoculated with each isolate and incubated
at 4, 10, 15, 20, 25, 30, 35, or 40°C.
included as a reference.
Vibrio anguillarum was
At intervals, the optical density at
525 tim was measured using a Bausch and Lomb Spectronic 20.
24
Growth
,
Different Concentrations j Sodium Chloride
Selected isolates (NZ, CHU, Ku-2, and Ku-4) were tested for
their ability to grow in different concentrations of sodium
chloride.
Tryptone glucose yeast broth (TGY) consisting of 1.0%
tryptone (Difco), 0.5% yeast extract (Difco), and 0.25% glucose
(Difco), was supplemented with sodium chloride to give concentrations of 0, 0.5, 1.5, 3, 4.5, 6, 8, and 10%.
Tubes containing 5
ml of TYG broth were inoculated in replicate and incubated at
Growth was determined by reading the optical density (525
18°C.
nm) at selected intervals.
Serological Tests
Production j Rabbit Antiserum
Antisera used in serological tests were prepared against NZ,
Cells were grown on spread plates
CHU, Ku-2, and Ku-4 isolates.
of BHI agar incubated at 18°C.
strain.
Four plates were used for each
When the cultures were confluent (48-72 h), the cells
were removed and washed three times with sterile PBS (pH 7.0) by
centrifugation (3,840 x g, 20 mm
at 4°C).
The cell pellets were
resuspended in 6-7 ml PBS and heated at 100°C for 1.5 h to
destroy flagellar antigens.
The heated cells were then pelleted
by centrifugation, washed twice in PBS and resuspended in 5-10 ml
PBS.
Four New Zealand white rabbits were used for antiserum
production.
Each rabbit received intradermal injections of an
emulsion of antigen and an equal volume of Freunds complete
25
adjuvant in the hind foot pads (0.05 ml each pad) and at two
sites between the scapula (0.1 ml at each site).
One month following the initial injection, a booster
injection containing 1.0 ml of cell suspension and Preunds
incomplete adjuvant was given intradermally at five sites between
the scapula.
Before the booster injection and at weekly
intervals thereafter, 50 ml of blood was drawn from the marginal
ear vein of each rabbit.
The blood was allowed to clot at room
temperature for two hours and then placed at 4°C overnight for
clot retraction.
After centrifugation (1520 x g for 20 mm), the
serum was harvested, filter sterilized using a membrane filter
(0.45 micron pore size, Gelman Sciences Inc.), and frozen in
sterile tubes.
Antisera against
.
anguillarum (LS-173) and
ordalii
(MSC-275) were obtained from Dr. Robert E. Olson, Oregon State
University.
They had been prepared by injecting emulsions of
live bacteria and Freund's adjuvant intradermally at 15 Sites as
described by Handoyo (1985).
Slide Agglutination Tests
Antisera against
.
anguillarum,
. ordalii, NZ, CHU, Ku-2,
and Ku-4 were tested for cross-agglutination of all nine isolates
by rapid slide agglutination.
Each bacterial antigen was
prepared as heat-killed antigen and whole cell antigen.
The heat-
killed antigens were prepared in the same manner as the antigens
used for injecting rabbits except that the bacterial suspensions
were adjusted with PBS to an optical density of 1.0 at 525 nm.
26
The whole cell antigens were grown on TSA and harvested between
48-72 h.
The cells were washed three times in PBS and adjusted
to an optical density of 1.0 at 525 nm.
One drop of each antigen to be tested was added to one drop
of appropriate antiserum in a concavity slide.
A well containing
one drop of antigen added to one drop of PBS was used as a
control for auto-agglutination.
The slides were gently rotated
for 2-3 miii to mix the suspensions.
Agglutination was determined
bothmacroscopically and microscopically using a light box and a
dissecting microscope.
Determination j Agglutinating Antibody Titer
The agglutinating antibody titers of the rabbit antisera
were determined using a microtiter system (Cooke Engineering
Co.).
Antigens for use in this experiment were prepared in the
same manner as the antigens used in slide agglutination tests
except that the optical density was adjusted with PBS to an OD
(525 nm) of 0.85.
One hundred microliter aliquots of antiserum
were placed in the first row of wells of a round bottom
microtiter plate and serial two-fold dilutions were made in PBS
using 0.05 ml micro-diluters (Cooke Engineering Co.).
Controls
with no antiserum were used in each plate to detect auto-
agglutination. An equal volume of antigen was added to each
well.
The plates were covered to prevent evaporation, incubated
for 2 h at room temperature and then overnight at 4°C.
The
dilution of the last well that displayed agglutination was
recorded as the titer of the antisera.
27
Ouchterlonv Double Diffusion Tests
To determine antigenic relationships among the isolates
studied and reference strains,
.
anguillarum and
ordalii,
Ouchterlony double diffusion plates were prepared using antisera
against
. anguillaruin,
.
odalii, NZ, CHU, Ku-2, and Ku-4.
The
plates were made using 0.8% agarose in borate buffered saline (5
parts borate buffer/95 parts 0.85% NaC1 in distilled water).
The
buffer consisted of 6.184 g boric acid, 9.536 g borax, and 4.384
g NaC1 per liter of deionized distilled water.
The plates were
precoated with 15 ml of 0.3% aqueous agar and placed in an 80°C
oven until thoroughly dry leaving a thin film of agar on the
bottom of the plate.
Hot agar was then added to a depth of
approximate 2-3 mm and allowed to harden.
Wells were cut in the
desired pattern and excess gel was withdrawn by low vacuum.
Antigens were prepared by inoculating each isolate into
200 ml BHI broth which was then incubated at 18°C for 48-72 h
The culture was harvested and washed three times in PBS.
The
cells were then resuspended in a minimal volume (2-5 ml) of PBS
and sonicated until they were disrupted as determined by phase
contrast microscopy.
Antigens and antiserum were added to the
wells and the plates were incubated in a humid chamber.
After
the precipitation bands had developed, photographs were taken
using a light box and Kodak technical pan film 2415.
28
Percent Guanine Plus Cytosine Determination
Isolation of DNA
To conduct %G+C analysis, it was necessary to extract deoxy-
ribonucleic acid (DNA) from each isolate.
The techniques used
for DNA isolation were those described in the Manual of Methods
General Bacteriology (Johnson, 1981).
These represent a
modification of the method of Marmur (1961).
Three grams of wet,
packed cells were centrifuged and washed three times in salineEDTA (O.15M NaCl, O.O1M sodium-EDTA, pH 8.0).
The cells were
resuspended in 50-100 ml saline-EDTA, and disrupted by the
addition of 20% (W/V) sodium dodecyl sulfate (SDS) to a final
concentration of 1%.
The cell suspension was incubated in a
water bath at 50-60°C to increase the rate of lysis.
Cell lysis
was apparent by an increase in the viscosity of the solution and
change of the suspension from turbid to opalescent.
Dissociation
of protein from nucleic acid in the solution was performed by
adding 5M sodium perchlorate stock solution to a final concentration of 1M.
Deproteinization was carried out by addition of
an equal volume of 24:1 (V/V) chloroform-isoamyl alcohol; the
entire contents were then shaken on an agitator for 30 mm
centrifuged (11,200 xg, 10 mm) to separate the phases.
and
The
upper aqueous layer was carefully collected using an inverted 10
ml serological pipette with the tip inserted into a pipet-aid
(Drummond Scientific Co.).
An equal volume of chloroform-isoamyl
alcohol was added to the aqueous phase and the same procedures of
shaking, centrifuging, and collecting were repeated.
29
After 2-3 cycles of deproteinization, the upper aqueous phase
was collected and slowly overlayed with two volumes of cold 95%
ethanol to precipitate the DNA.
The DNA strands were gently
spooled around a glass rod and dissolved in 10-20 ml of dilute
saline citrate (O.1XSSC: 0.015M NaC1, 0.0015M trisodium citrate).
After the DNA was completely dissolved, 2OXSSC was added to
adjust the concentration to 1XSSC.
Bovine pancreatic RNase
(Sigma Chemical Co.) was prepared as a stock solution (1 mg/mi in
to inactivate any
0.15M NaC1, pH 5.0. heated at 80°C for 10 mm
traces of DNase) and added to the DNA solution to a concentration
of 0.05 mg/mi.
The mixture was incubated at 37°C for 30 mm.
Deproteinization and DNA precipitation were repeated to remove
free ribonucleotides. Finally, the isolatI DNA was dissolved in
0.1XSSC and stored at -20°C.
Determination of Percent Guanine Plus Cytosine
To determine the percent guanine plus cytosine of NZ, CHU,
Ku-2, and Ku-4 isolates, thermal melts (Tm) were performed using
Escherichia coli, strain WP2 as a reference.
DNA from the four
selected isolates was prepared as described.
Reference DNA from
E.. coli, strain WP2 was obtained from Richard A. Holt, Oregon
State University.
in O.1XSSC.
The DNA preparations were dialyzed overnight
Thermal melts (Tm) were performed using a Beckman
model DU-8 spectrophotometer equipped with a Tm Compuset Model.
Each thermal melt contained a reference
coli WP2 DNA cell,
four cells with vibrio DNA, and a O.1XSSC cell.
done in duplicate.
Each test was
The DNA was added to the cuvettes at a final
30
concentration of 25-50 ug/ml.
Absorbance readings were corrected
for thermal expansion and melting temperatures were determined.
The percent guanine plus cytosine (%GC) was calculated by
the equation of Mandel et al (1970):
%GCX = %GCstd+ 1.99 (Tm- Tnlstd)
where %GCX is the %GC of the tested isolate.
The %GCstd for L.
coli WP2 used as a standard was 51 mol% guanine plus cytosine
(Seidler and Mandel, 1971).
Virulence
Patho2enicity Tests
Injection Challenge
Experimental infection was performed in fingerling rainbow
trout injected with NZ, CHU, Ku-2, and Ku-4 cells.
The four
isolates were grown in TSB at 18°C, harvested in log phase, and
washed three times with PBS.
Ten fold serial dilutions of each
culture were made in PBS, and plate counts were performed to
determine the number of viable cells.
Ten rainbow trout (mean Vt
4.18 g) per dilution were injected intraperitoneally (i.p.) with
0.1 ml of the test dilution and held in an aquarium containing
five liters of water.
The water temperature was maintained
Control fish were injected with 0.1 ml of PBS.
between 19-20°C.
Dead fish were collected daily and necropsied to ascertain cause
of death.
LD50s were calculated by the method of Reed and
Muench (1938).
In addition, mean day to death (MDD) was calcu-
lated using the equation:
MDD
=
Z(numbers of deaths) (day of death)
total number of dead fish
31
Waterborne Challenge
Pathogenicity of isolates Ku-2 and Ku-4 was also tested by
waterborne challenge using chinook salmon (mean Vt 51.8).
Procedures were modified from the method described by Handoyo
(1985).
Static water aquaria (300 liters) provided with aeration
were used for these exposures.
Ten fish were placed in each
aquarium and the water level lowered to 40 L.
Three amounts (10
ml, 100 ml, and 1000 ml) of TSB cultures were harvested in log
phase and added to replicate aquaria.
After a 30 mm
exposure,
water was restored to the normal level with a flow rate between
2-3 L/min and the temperature maintained between 18-19°C.
Control fish were treated in the same manner by additional 1000
ml uninoculated TSB.
Triplicate plate counts were made to
enumerate the number of bacteria in each treatment.
were collected daily and examined for cause of death.
day to death was calculated as above.
Dead fish
The mean
32
RESULTS
The isolates used in this study (NZ, CHIT, Ku-i, Ku-2, Ku-3,
Ku-4, Ku-5, Ku-6, and Ku-i) possessed morphological, physiologi-
cal, and biochemical characteristics of members of the genus
Vibrio.
Microscopic examination of bacterial cells grown in
tryptic soy broth (TSB) showed the organisms to be straight or
curved rods, ranging from
1.2-2.9 urn in length and 0.6-1.1 urn in
width (measured after Gram-stain).
All isolates were Gram-
negative; motile; cytochrome oxidase positive; ferinentative
for glucose; and sensitive to the vibriostatic compound, 0/129
(2 ,4-diamino-6,7-diisopropylpteridine).
The isolates studied
were phenotypically (Table 3) differentiated from each other and
from 20 recognized Vibrio species listed in Bergevs Manual
Systemic Bacteriology (Baumann et al., 1984).
None of the
isolates could be assigned to one of the established Vibrio
species, including nine reported to be pathogenic for fish (Table
1).
Growth Characteristics
Growth Curves
Mean Generation Times
Growth curves of the NZ, CHIT, Ku-2, and Ku-4 isolates were
determined using TSB incubated at 18°C.
Graphs were constructed
by plotting the optical density at 525 urn versus the viable cell
Table 3.
Phenotypic characteristics of Vibrio isolates used in this study compared with
anguillarum and . ordalii.
.
Isolates
Characteristics
LS-i74 MSC-275
Motility
Gramstain
PHBaccumulation
-D-galactosidase (ONPG)
Arginine dihydrolase
Lysine decarboxylase
Ornithine decarboxylase
Citrate utilization
H2Sproduction
Urease
Tryptophane deaminase
Indoleformation
Voges-Proskauer reaction
Gelatinase
Utilization of:
Glucose
Mannitol
Inositol
Sorbitol
Rhamnose
Sucrose
Melibiose
Amygdalin
Arabinose
NZ
+
+
+
-
+
+
+
+
-
+
(+)
(+)
+
+
+
(+)
+
+
+
-
+
(+)
(+)
(+)
-
+
-
CHU
+
Ku-2
+
-
+
+
+
+
+
+
-
-
+
+
-
Ku-4
+
+
+
+
-
Ku-i
+
+
+
-
Ku-3
+
-
-
Ku-5
(-i-)
-
-
Ku-7
+
+
-
+
+
+
+
+
+
-
+
+
-
+
-
(+)
+
+
-
Ku-6
+
-
+
+
+
+
-
+
-
-
+
-
-
+
+
+
+
+
-
+
-
+
+
+
+
-
-
-
-
-
-
+
-
+
-
Table 3.
(Continued)
Isolates
Characteristics
Cellobiose
D-Gluconate
Gas from D-glucose
Reduction of NO'to NO
Lipase
Cytochrome Oxidase
SensitivitytoO/129
Pigmentation
Swarming on solid media
Growth at:
4°C
30°C
35°C
40°C
+
+
+
+
+
+
+
+
+
+
+
-
= Positive reaction
= Negative reaction
(+) = Weak positive reaction
+
-
CHU
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
-
-
+
-
+
-
-
+
+
+
-
0%NaC1
3%NaC1
6%NaC1
8%NaC1
i0%NaC1
Ku-4
NZ
LS-174 MSC-275
+
-
+
+
-
Ku-2
Ku-i
Ku-3
Ku-5
Ku-6
+
+
Ku-7
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
+
+
+
+
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
-
-
+
-
-
-
+
-
+
+
+
+
35
number per milliliter (Figure 1, 2, 3, and 4).
Mean generation
times for NZ, CHU, Ku-2, and Ku-4 were calculated by the method
of Stanier et al. (1970).
At 18°C these values were 0.68, 0.49,
0.82, and 0.87 h, respectively.
Growth at Different Temperatures
Growth rates at selected temperatures were determined for
the
.
anguillarum, NZ, CHIJ, Ku-2, and Ku-4 isolates.
At
intervals, the OD at 525 tim was determined using TSB media
incubated between 0-40°C (Figure 5, 6, 7, 8, and 9).
CHU, HZ,
Isolates
anguillarum, Ku-2 and Ku-4 grew best at 20, 25, 25,
35, and 35°C, respectively.
at 30°C or higher.
The CHIJ isolate was not able to grow
The HZ and
.
anguillarum isolates were
inhibited at 35 and 40°C, respectively.
The Ku-2 and Ku-4
isolates grew more rapidly at the higher temperature (40°C) than
at the lower ones (10 and 15°C).
Growth
Different Concentrations j Sodium Chloride
To determine the effect of various concentrations of NaCl on
the growth of HZ, CHU, Ku-2, and Ku-4, and 1. anguillarum, growth
curves plotting the optical density at 525 nm versus incubation
times of each organism were constructed (Figure 10, 11, 12, 13,
and 14).
All organisms displayed a requirement for NaC1.
The
HZ, CHU, Ku-2, and Ku-4 isolates did not grow in the absence of
NaCl (0% Had); however, all grew when NaC1 concentrations were
increased (0.5-4.5% NaC1).
The optimum concentrations of NaC1
for these isolates were between 1.5-4.5%.
The four selected
36
1
10
1
o8
a,
U
a,
.a
a,
106
io4J
I
0
2
1
3
Optical Density (525 nm)
Figure 1
Growth curve of NZ isolate in tryptic soy
broth at 18 C plotting viable cells /ml
versus optical density.
101
1 0
15
io8
E
a
a,
'U 7
a,
15
106
1
0
1
2
Optical Density (525 nm)
Figure 2
Growth curve of CHU isolate in tryptic soy
broth at 18 C plotting viable cells /ml
versus optical density.
37
1011
1010.
U
1o8
U.
1
io6T
0
3
2
1
Optical Density (525 nm)
Figure 3 Growth curve of Ku-2 isolate in tryptic
soy broth at 18 C plotting viable cells /ml
versus optical density.
1010
U
1
E
io
1
1 o6
0
2
1
3
Optical Density (525 nm)
Figure 4
Growth
curve
of Ku-4
isolate
in
tryptic
soy broth at 18 C plotting viable cells /ml
versus optical density.
38
1.0
0.8
E
-a- bc
It)
..- 15C
c'J
It)
- 200
-0-a-0-0-
0)
C
C)
0.4
C)
U
250
30C
350
400
C).
0
0.2
0.0
u
I
.
30
20
10
0
I
I
I
I.
50
40
hours
Figure 5
Growth of the NZ isolate in tryptic soy
broth incubated at different temperatures.
0.8
E
C
0.6
It)
-a- 100
-.- 15C
U)
-0- 250
U)
Csl
- 200
30C
C,
. 35C
0
U
o
C).
-0- 400
0.2
0.0
I
0
.
I
10
I.
30
20
.
40
UI
50
hours
Figure 6 Growth of the CHU isoiate in tryptic soy
broth incubated at different temperatures.
39
1.2
1.0
E
It)
c.1
It)
bC
0.8
15C
- 20C
0.6
25C
30C
35C
40C
w
U
0.
0
o.o-t.--v-1
-:
10
0
.
30
20
40
50
hours
of the Ku-2 isolate in tryptic
soy broth incubated at different tem-
Figure 7 Growth
peratures.
1.0.
I
0.8
E
-0- 1OC
.4- 15C
It)
It)
- 20C
.4- 25C
C
- 30C
0.4
-0- 35C
-*- 40C
U
0
0.2
0.0-t
10
0
20
30
40
50
hours
Figure
8
Growth of the Ku-4 isolate in tryptic
soy broth incubated at different temperatures.
40
1.0
0.8
E
0- 1OC
In
a
.4- 15C
0.8
- 20C
>
-0- 25C
..- 30C
-0- 35C
CD
CD
0.4
4- 40C
CD
C.)
0.2
0.0-t
10
0
30
20
40
50
hours
Figure 9 Growth of Vibrio anguillarum in tryptic
soy broth incubated at different temperatu res.
0.5
0.4
E
-0- 0%
IC)
-.- 0.5%
-U- 1.5%
C..J
In
-0- 3%
U)
- 4.5%
0.2
-0- 6%
-0- 8%
CD
C.)
6- 10%
a.
0
0.1
0.0
0
'
.
I
10
30
20
40
I
50
hours
Figure 10
of the NZ isolate in tryptone
glucose yeast broth at different con-
Growth
centrations of NaCI incubated at 18°C.
41
0.4
0.3
- 0%
..- 0.5%
-U- 1.5%
LI)
c.l
It)
-.- 3%
(5
- 4.5%
15
-0- 6%
-e- 8%
-6- 10%
(5
o
C.
0.1
0.0
I
--çBB-.
10
0
i
40
30
20
50
hours
Growth of the CHU isolate in tryptone
glucose yeast broth at different concentrations of NaCI incubated at 18°C.
Figure 11
0.5
0.4
-0- 0%
U)
-.- 0.5%
-U- 1.5%
C'4
II,
-0- 3%
(5
C
- 4.5%
0.2
-0- 6%
-6- 8%
-6- 10%
15
U
0
C.
0.1
0.0J
I-BM.
0
10
.
20
.
30
I
40
50
hours
Figure 12
Growth of the Ku-2 isolate in tryptone
glucose yeast broth at different concentrations of NaCI incubated at 18°C.
42
0.6
0.5
E
-D- 0%
4- 0.5%
.- 1.5%
.4- 3%
- 4.5%
-0- 6%
-4- 8%
.4- 10%
E
I
0.1
0.0
I
-!--M
0
10
.
20
I
.
30
40
50
hours
Figure 13
Growth of the Ku-4 isolate in tryptone
glucose yeast broth at different concentrations of NaCI incubated at 18°C.
0.8
E
0.6
-0- 0%
.4- 0.5%
-I- 1.5%
It)
C4
It)
.4- 3%
fi,
o
- 4.5%
-a- 6%
-4- 8%
-6- 10%
0.2
0.01
0
10
20
30
40
50
hours
Figure 14
Growth of Vibrio anguillarum
tryptone glucose yeast broth at different
concentrations of NaCI incubated at 18°C.
in
43
isolates grew best at 3% NaCl, but
1.5% NaCl.
.
anguillarum grew best at
All isolates tested grew more slowly at NaCl
concentrations greater than 4.5%.
All were inhibited at NaC1
concentrations of 8 and 10%.
Serolo2ical Tests
Slide Agglutination Tests
Antisera against
.
anguillarum,
.
ordalii, NZ, CHU, Ku-2,
andKu-4 were tested for their ability to agglutinateantigens
from all isolates used in this study.
The six antisera reacted
strongly with homologous antigens and slight agglutination was
observed with some heterologous antigens (Table 4).
None of the
antisera showed strong agglutination with the heterologous
antigens, indicating that all isolates were antigenically
distinguishable.
Determination j Alutinating Antibody Titers
The agglutinating antibody titers of rabbit antisera against
.
anui1larum,
.
ordalii, NZ, CHU, Ku-2, and Ku-4 were
used to determine the antigenic relatedness of nine new isolates.
All antisera exhibited the highest titers when reacted with
homologous antigens and lower titers when reacted with heterologous antigens (Table 5).
Although some antisera had relatively
high titers with heterologous antigens, all of the isolates were
antigenically distinguishable.
The differences in agglutinating
antibody titers observed between the nine new isolates were at
Table
4.
Results of slide agglutination tests using rabbit antisera against Vibrio anguillarum, V. ordalii, NZ, CHU, Ku-2, and Ku-4 reacted with homologous and heterologous antigens.
Antigens
Antisera
LS-174
MSC-275
NZ
Ku-i
Ku-2
Ku-4
s
-
s
s
s
-
-
$
-
-
-
s
s
-
-
CHU
Ku-3
Ku-5
Ku-6
Ku-7
LS-174
+
$
MSC-275
8
+
-
NZ
-
-
+
-
-
-
5
-
S
-
-
CHU
-
s
-
+
-
-
-
-
-
-
-
Ku-2
-
-
-
-
+
-
-
S
-
-
-
Ku-4
-
-
-
-
-
+
-
-
s
-
-
+ = Heavy agglutination
s = Slight agglutination
- = Non-agglutination
Table 5.
Agglutinating antibody titers of rabbit antisera against Vibrio anguillarum. 1.
ordalii, NZ, CHU, Ku-2, and Ku-4 when reacted with the homologous and heterologous antigens.
Titer when tested with antigens
Ant is era
LS-174
LS-174
NSC-275
NZ
CHU
Ku-2
Ku-4
Ku-i
Ku-3
Ku-5
Ku-6
Ku-i
i28
16
8
8
8
8
8
8
8
8
8
32
128
8
16
4
8
4
16
16
8
8
NZ
2
16
265
8
4
8
64
32
64
2
2
CR11
8
32
16
128
32
8
8
16
16
8
8
Ku-2
8
8
2
8
1032
8
16
128
16
32
2
Ku-4
4
4
<2
4
4
64
8
8
8
2
2
MSC-275
U,
46
least as great as the cross-agglutinating titers between L
anguillarum and
ordalii suggesting the nine isolates belong,
at least, to separate serotypes.
Ouchterlonv Double Diffusion Tests
Vibrio anguillarum,
.
ordalii, NZ, CHU, Ku-2, and Ku-4
antisera were diffused against sonicated cell extracts of each of
the nine new isolates,
15A-L).
.
anguillarum, and VL. ordalii (Figure
In homologous reactions, the antisera recognized at
least three antigens forming obvious precipitin bands.
the antisera against
. anguillarum and
.
Because
ordalii were prepared
against whole cells, they formed 1-4 additional cross-precipitin
bands with antigens from all isolates.
The antisera against the
thermostable (0) antigens of NZ, CHU, and Ku-2 also cross-precipitated some heterologous antigens; in most cases, only one
cross-precititin band was formed.
The NZ and CHU antisera
recognized a common antigen among the Ku-i and Ku-5 isolates.
A "pattern of identity" (Oudin and William, 1971) in which the
precipitin bands coalesced at the end forming a continuous precipitin band was observed (Figure 15F and 15H).
Percent Guanine Plus Cytosine Determination
Percent guanine plus cytosine of HZ, CHU, Ku-2, and Ku-4
was determined using thermal melts (Tm) and calculated by the
equation of Mendel et al. (1970).
The average percent guanine
plus cytosine (%G+C) of NZ, CR13, Ku-2, and Ku-4 isolates were
47
Figure 15.
Ouchterlony double diffusion plates. Antigens in the
outer veils were reacted with the following antisera
in the center wells:
Plate A.
Vibrio anguillarum, strain LS-174
Plate B.
Vibrio anguillarum, strain LS-174
Plate C.
Vibrio ordalii, strain MS-275
Plate D.
Vibrio ordalii, strain MS-275
Plate E.
Isolate NZ
Plate F.
Isolate NZ
Plate G.
Isolate CHU
Plate H.
Isolate CHU
Plate I.
Isolate Ku-2
Plate J.
Isolate Ku-2
Plate K.
Isolate Ku-4
Plate L.
Isolate Ku-4
- is
LS
174
NZ
Ku4
Ku3.
Ku
Anti
Anti
15174
15-174
Ku ô
Ku2.
',Ku-5
EE
MSC
k
275
15
174
1)
-
MSC
275
Ku 4
Ku 3
\\Z7Y
CHU
Ku2
MSC
Ku-6
Ku 5
Kul
-
275
Ku 4
Ku-7
NZ)
(.__)
CHU
Ku-S
"1
Ku2
Ni
C
e
L27)
Ku-2
Anti
174
IS
275
IMsc
Ku-4
0.
.1.1
.
Ku-3
Ku6
.f_
.
Ku'4
Anti
Ku-2
Ku2
Ku-7
0
Ku 7
-
44.7, 38.1, 43.8, and 41.8, respectively (Table 6).
These
values fell within the 38 to 51 %G+C value reported for the genus
Systemic Bacteriology (Baumann et
Vibrio in Bergeys Manual
al., 1984).
The differences in %G+C among these four isolates is
as great as the differences among most of the species of Vibrio
suggesting these isolates are genetically diverse.
Virulence
Injection ChaiJenge
.
Pathogenicitv Tests
Fifty Percent Lethal Dose
To compare the virulence of the NZ, CRU, Ku-2, and Ku-4
isolates, experimental infections were produced in fingerling
rainbow trout (mean wt. 4.18 g).
The fish were injected
intraperitoneally with selected numbers of bacterial cells ranging
from 1.5 x 1O4 to 1.5 x 1O9 NZ cells, 2.8 x 1O4 to 2.8 x 1O9 CHU
cells, 2.1 x
io
to 2.1
Ku-4 cells.
10
Ku-2 cells, and 1.5 x 1O4 to 1.5 x
The percent mortalities of fish caused by the
organisms ranged from 0 to 100% with a mean day to death of 1-6.5
days.
The fifty percent lethal dose (LD50) of the NZ, CHU, Ku-2,
and Ku-4 isolates, as calculated by the method of Reed and Muench
(1938), was 7.2 x io8 cells, 2.8 x io8 cells, 1.4 x io6 cells,
and 1.3 x io8 cells, respectively (Table 7, 8,
9, and 10).
Generally, no internal or external pathological signs were
observed in fish that died during the first two or three days
post injection; however, infectious organisms were recovered from
kidney smears cultured on tryptic soy agar and identified by API20E strips.
Diseased fish dying three or more days post injec-
51
Table 6.
Isolate
Percent guanine plus cytosine (%G+C) values of the NZ,
CHIJ, Ku-2, and Ku-4 isolates using Escherichia coil
(WP2) as the reference strain.
%G+C
Average %G+C
NZ
44.70
44.75
44.7
CHU
38.06
38.04
38.1
Ku-2
43.31
44.19
43.8
Ku-4
41.78
41.82
41.8
52
Table 7.
Number of
cells
injected
Mortality and mean day to death of rainbow trout
injected with different numbers of NZ ceilsa.
Number of
fish
injected
Number of
deaths
% Mortality
Mean day
to death
1.5 x
10
6
60
1.2
1.5x108
10
2
20
1
1.5x107
10
0
0
1,5x106
10
0
0
1.5x105
10
0
0
1.5x104
10
0
0
0.1M PBS
10
0
0
-
a
LD50 calculated by the method of Reed and Muench (1938) was
7.2 x 1o8 cells.
53
Table 8.
Mortality and mean day to death of rainbow trout
injected with different numbers of CHU cellsa.
Mean day
to death
Number of
cell
injected
Number of
fish
injected
2.8 x io
10
10
100
1
2.8 x io8
10
5
50
1
2.8x107
10
0
0
2.8x106
10
0
0
2.8x105
10
0
0
2.8x104
10
0
0
0.1M PBS
10
0
0
Number of
deaths
% Mortality
-
calculated by the method of Reed and Muench (1938) was
LD
2.8 x 1o8 cells.
54
Table 9.
Number of
cells
injected
Mortality and mean day to death of rainbow trout
injected with different numbers of Ku-2 cellsa.
Number of
fish
injected
Number of
deaths
% Mortality
Mean day
to death
2.1 x io
10
9
90
1
2.1 x io8
10
10
100
1
2.1 x 10
10
9
90
1.4
2.1 x 106
10
5
50
3.8
2.1
10
3
30
5.3
2.1x104
10
0
0
0.1MPBS
10
0
0
10
a
LDç0 calculated by the method of Reed and Muench (1938) was
1.4 x 1o6 cells.
55
Table 10.
Number of
cell
injected
Mortality and mean day to death of rainbow trout
injected with different numbers of Ku-4 celisa.
Number of
fish
injected
Number of
deaths
% Mortality
Mean day
to death
1.5x109
10
9
90
1
l.5x108
10
5
50
3
1.5 x 10
10
2
20
6.5
1.5x106
10
0
0
1.5x105
10
0
0
-
1.5x104
10
0
0
-
0.1M PBS
10
0
0
-
aLD
0
calculated by the method of Reed and Muench (1938) was
x 1o8 cells.
56
tion often had swollen abdomens, darkened skin, and were inactive.
Hemorrhagic areas were seen at the base of the fins, at the
vent, the lower parts of the opercula, within the mouth, and on
the body surface (Figure 16A-C).
When necropsied, ascites and
white membranous material were observed inside the body cavity
and around viscera and mesenteries.
and gut were most often affected.
The spleen, kidney, liver,
In heavily infected fish, the
affected organs were often deformed and/or liquidfied.
Waterborne Challenge
Because the Ku-2 and Ku-4 isolates demonstrated greater
virulence for rainbow trout than the NZ and CHU, further studies
of the Ku-2 and Ku-4 isolates were conducted using waterborne
challenges.
Juvenile chinook salmon (mean wt. 51.8 g) were
exposed to varying numbers of each isolate ranging from 5.7 x
lO-5.7 x 1O
cells and 1.3 x l0-1.3 x io
Ku-4, respectively.
cells of Ku-2 and
Mortalities caused by the Ku-2 isolate were
low, ranging from 0-15% with a mean day to death of 2.5-17.5 days
(Table 11).
The Ku-4 isolate mortalities ranged from 0-10% with
a mean day to death of 2.5-5.8 days (Table 12).
Generally,
pathological signs produced by waterborne challenge were similar
to the signs observed in fish challenged by injection; however,
the signs of the disease induced by waterbore challenge progressed more slowly.
In addition, severe skin lesions of Ku-2
diseased fish were often seen at the snout and around the caudal
peduncle (Figure 17A-B).
Pure cultures of the organism were
recovered directly from lesions.
57
Figure 16.
Characteristic hemorrhagic septicemia in rainbow
trout injected by isolates studied.
A.
Swollen abdomen and darkened skin
B.
Hemorrhaging at the base of fins, at the vent,
and the lover parts of opercula
C.
Hemorrhaging on the body surface
58
C
Figure 16.
59
Table 11.
Mortality and mean day to death of chinook salmon
waterborne challenged with Ku-2.
Number of
cells added
to the water
5.7 x 10
Replicates
1
2
Average
5.7x106
1
2
Average
5.7 x 1O
1
2
Number of
fish
exposed
10
10
-
20
10
10
-
0
10
10
1
2
Average
Mean day
to death
ID..
6.0
11.0
8.5
15
0
La1
2.3
10
18.0
17.0
17.5
10
10
Average
Controla
% Mortality
10
10
0
Q.
0
aControl fish were exposed to 1000 ml of tryptic soy broth.
-
60
Table 12.
Mortality and mean day to death of chinook salmon
waterborne challenged with Ku-4.
Number of
cells added
to the water
1.3 x 1O7
Replicates
1
2
Average
1.3x106
1
2
Average
1.3x105
1
2
Average
Controla
1
2
Average
Number of
fish
exposed
Z Mortality
10
10
-
20
10
10
-
0
Mean day
to death
5.0
Q.
Q.
2.5
10
0
10
11.5
5.8
10
10
-
0
10
10
-
0
Q.
0
Q.
0
aControl fish were exposed to 1000 ml of tryptic soy broth.
61
Figure 17.
External pathological signs in chinook salmon exposed
to Ku-2 isolate.
A.
Lesions at the snout
B.
Lesions around the cauda]. peduncle
62
A
Figure 17.
63
DISCUSSION
Nine isolates of bacteria, presumed to be members of the
genus Vibrio were obtained for this study.
Initial tests
involved morphological and biochemical analysis to confirm the
isolates were, in fact, vibrios.
From results obtained, four of
the isolates (NZ, CR13, Ku-2, Ku-4) were selected for additional
analysis: 1) NZ from southern hemisphere salmonids, 2) CR13 from
North American salmonids, 3) Ku-2 because it is the most common
type recovered from fish in Kuwait (V. Rasheed, personal commu-
nication), and 4) Ku-4 because it was the most different from
Ku-2.
Based on morphology and biochemical reactions, all nine of
the isolates studied, NZ, CR11, Ku-2, Ku-4, Ku-i, Ku-3, Ku-5,
Ku-6, and Ku-?, are members of the genus Vibrio as described in
Ber2evs Manual
Svstemic Bacteriolov (Baumann et al., 1984).
In addition, the percent guanine plus cytosine (%G+C) determined
for the NZ, CR13, Ku-2, and Ku-4 isolates (38.1-44.7) fell within
the values characteristic for the genus (38-51%).
All isolates were phenotypically different from each other
(Table 3), from vibrios known to be pathogenic for fish (Table
1), and from other named Vibrio species listed in Bergeys
Manual.
Because the isolates differed from the established
species in key biochemical tests used to separate vibrios, it
appears that the isolates used in this study represent new
species.
However, DNA hybridization and additional biochemical
tests will have to be performed to support this conclusion.
64
Among the isolates tested, the closest relationship with any
established species was seen with the Ku-2 isolate which shared
certain phenotypic characterisics with 3 alginolyticus.
The
Ku-2 isolate was different in having a negative Voges-Proskauer
reaction, in being unable to grow in the presence of 8 and 10%
sodium chloride, and in having the ability to utilize amygdalin
and cellobiose.
In addition, the %G+C of Ku-2 (43.8) was below
that listed for V. alginolyticus (45-47).
Among other Kuwait
isolates, Ku-i and Ku-5 shared most biochemical characteristics
except
Ku-i utilized sucrose but not lipid; whereas, Ku-5 could
use lipid but not sucrose.
These isolates also appeared to share
at least one common antigen.
All isolates displayed an absolute requirement for NaC1.
This requirement is one of the characteristics of vibrios.
Four
of the isolates tested, NZ, CRU, Ku-2, and Ku-4 grew best at 3%
NaC1.
This level is higher than the value obtained for 'L.
anguillarum (1.5% NaC1) and explains why L anguillarum can be
isolated on normal bacteriological media but the strains obtained
for this study were only recovered on media supplemented with
NaC1 at 1.5% or greater.
The optimum growth temperature of the isolates tested
differed.
The Ku-2 and Ku-4 isolates grew best at 35°C whereas
the New Zealand (NZ) and chum salmon (Cml) isolates were inhibited
at 35 and 30°C growing best at 25 and 20°C, respectively.
This
was consistent with the temperature optima of the hosts from
which the isolates were obtained.
Based on the results of Ouchterlony double diffusion, slide
65
agglutination, and microtiter agglutination tests, the NZ, CHU,
Ku-2, and Ku-4 isolates possessed unique thermostable (0)
antigens specific for each isolate.
This was indicated by
formation of strong precipitin bands in double diffusion plates
and heavy agglutination when reacted with the homologous antigens
in slide agglutination or microtiter assay.
Although the
antisera against HZ, CHU, and Ku-2 cross-precipitated with some
heterologous antigens in Ouchterlony plates, the observed bands
were different from the bands formed in homologous reactions.
In
most cases, only one faint cross-precipitin band was formed.
This was assumed not to be a reaction with the major 0 antigen.
Lipopolysaccharides (LPS) represent the major 0 antigens in the
vibrio-like bacteria pathogenic for fish (Chart and Trust, 1984).
These LPS generally form obvious dense precipitin bands with
homologous antisera (S. Kaattari, personal communication).
To
further investigate the major 0 antigens of each isolate,
extracted and purified LPS from each organism should be used.
In
addition, antigenic relationships based on minor antigens of each
isolate should be determined.
This could be performed by cross
adsorption studies and by placing the common cross-precipitating
isolates adjacent to each other in wells of an Ouchterlony plate.
Several additional precipitin bands were formed in
Ouchterlony plates when the isolates were reacted with
anzuillarum and
.
.
oIalii antisera, probably because these
antisera were prepared against whole-cells.
The antisera against
HZ, CHU, Ku-2, and Ku-4 were prepared against heat-killed antigen
preparations and possessed antibodies specific only to
66
thermostable (0) antigens.
Results from Ouchterlony double diffusion plates also demon-
strated the existance of at least one common antigen between the
Ku-i and Ku-5 isolates as indicated by the formation of a band of
identity on each of two plates (Figure 15F, 15H).
This antigenic
identity along with extensive phenotypic similarity and the fact
that both organisms were isolated from tilapia at the same location suggests that the Ku-i and Ku-5 isolates are closely related
and probably members of the same species.
Experimental infection produced in fingerling rainbow trout
injected with various numbers of NZ, CHU, Ku-2, and Ku-4 cells
induced a characterisic Gram-negative hemorrhagic septicemia.
No external or internal pathological signs were observed, however, in fish that died within 2 or 3 days post injection.
It is
likely that in these acute infections, fish died before lesions
and extensive pathology could develop (Handoyo, 1985).
The lower
LD50 values obtained for Ku-2 and Ku-4 indicated that these
isolates had greater virulence for rainbow trout than the CHU and
NZ isolates.
The virulence and pathogenicity of Ku-2 and Ku-4
were further tested in chinook salmon using waterborne challenge.
The signs of disease produced by Ku-2 and Ku-4 in chinook salmon
progressed more slowly in these fish than by i.p. challenge in
rainbow trout as indicated by the longer mean day to death.
The
differences in mean day to death in these tests are difficult to
compare because the size and species of fish used in injection
and waterborne challenges were different.
Using chum salmon and
English sole, (Parophrys vetulus) to compare pathogenicity of
67
anguillarum by intraperitoneal injection and waterborne challenges, Handoyo (1985) reported a significantly higher mean day
to death following waterborne challenge in both species of fish.
Although Ku-2 and Ku-4 were originally isolated from nonsalmonid fish, they were able under experimental conditions, to
infect salmonids.
In addition, the Kuwait isolates exhibited
greater virulence for rainbow trout than the NZ and CHU isolates
which were originally recovered from salmonids.
growth temperature for Ku-2 and Ku-4 was 35°C.
The optimum
It is assumed
that if water temperature was increased, higher mortality would
be induced.
These data suggest that Ku-2 and Ku-4 could be high-
ly pathogenic for rainbow trout reared at near maximum temperatures.
While these isolates were not tested in warinwater spe-
cies, Ku-2 represents the type of vibrio most commonly isolated
from mortalities in tilapia reared in Kuwait.
The CHU and NZ isolates were less pathogenic in these tests;
however, under experimental conditions some vibrios isolated from
fish do not produce disease (Austin and Austin, 1987).
Several
factors may affect pathogenicity in fish including stress, water
temperature, salinity, water quality, nutrition, susceptibility
of fish species, and attenuation of virulence after several
passages or subcultures on artificial media.
In addition, a
pathogen associated with a disease may actually be a secondary
invader and require an external insult or infection to give entrance to the host.
Further pathogenicity tests using different
species of fish and different water temperatures will be required
to understand more about the virulence of the isolates.
In addi-
68
tion, back passage of these isolates in fish should be made to
test for increased virulence.
The taxonomy of the genus Vibrio is highly complex.
Several
classification systems have been used to describe vibrio-like
bacteria.
Generally, these were based on phenotypic characteris-
tics (Smith, 1961; Hastein and Smith, 1977; Kaper et al., 1983).
In contrast, several vibrios isolated from fish which were phenotypically different, have been classified as separate serotypes
of
.
an2uillarum on the basis of 0 antigen typing (Ezura et al.,
1980; Sorensen and Larsen, 1986; Tajima et al., 1986b).
Further-
more, based on phenotypic and/or serological characterizations,
several vibrio-like bacteria which have been isolated from
different species of fish in different geographic areas, are yet
unclassified (Mccarthy, 1976; Yasunaga and Yamamoto, 1977; Ransom,
1978; Kent, 1982).
The taxonomic status of vibrios pathogenic
for fish may be further complicated as a result of a study by
MacDonell and coiwell (1985).
Based on ribonucleotide sequence
determination of 5S rRNAs, the authors suggested a reclassification of y. anzuillarum and
.
damsela into a new genus,
Listonella.
The vibrio isolates used in this study demonstrated great
diversity phenotypically, serologically, and genetically (%G-i-C).
This diversity not only causes difficulty in taxonomy, it
suggests difficulty for control of vibriosis in mariculture as
well.
Protection of fish against vibriosis or other Gram-
negative bacteria by vaccination involves several factors.
the most important are antibodies against thermostable (0)
Among
69
antigens present on the outer membrane of these bacteria (Joklik
et al., 1980).
In some areas such as Pacific Northwest where
only a few serotypypes of vibrio cause losses in fish, vaccines
are coinmercailly available and effective.
However, in areas such
as Kuwait where numerous serotypes exist, if all are of equal
virulence, vaccination against these multiple serotypes appears
unlikely to provide good protection.
Because of the diversity among the many isolates of vibrios
from fish, it will be difficult to develop a simple classification scheme for these bacteria.
Classification methods based on
analysis of genetic material are becoming widely used and the
results are generally meaningful.
The techniques often used
include DNA/DNA and DNA/RNA hybridization, 16S rRNA oligomer
cataloging, and 5S rRNA sequencing.
Along with standardization
of an improved classification method to include genetic analyses,
additional vibrio isolates from different geographic areas and
different species of fish should be compared.
To gain understanding about the pathogenicity of vibrios,
research is needed to determine the effects of various factors on
the development of disease.
The factors should include water
temperature, salinity, and testing in the species of host from
which bacteria are isolated.
The mechanism of bacterial invasion
should also be investigated, especially for strains which do not
seem to be primary pathogens.
Finally, a more careful assessment of antigenic structure is
required.
This would assist in the development of additional
vaccines, a major tool for control of vibriosis in mariculture.
70
These vaccines should be able to induce cross protection among
the various vibrio isolates.
This may be achieved when we under-
stand more about how fish respond to various antigens.
With
further advances in genetic engineering, vaccines composed of
combinations of various major antigens which induce cross protection in fish could be produced.
71
SUMMARY AND CONCLUSIONS
1.
The isolates used in this study possessed morphological, physiological, and biochemical characteristics of the members of
the genus Vibrio.
2.
The isolates differed phenotypically from each other, from
vibrios known to be pathogenic for fish, and from other named
Vibrio species listed in Bergevs Manual
Systematic Bac-
teriolo2v.
3.
The percent guanine plus cytosine values obtained for the NZ,
CHU, Ku-2, and Ku-4 isolates (38.1-44.7%) were typical for
members of the genus Vibrio.
4.
All isolates displayed'an absolute requirement for NaC1.
Four of the isolates (NZ, CHU, Ku-2, and Ku-4) grew best in
media suppplement with 3% MaCi.
5.
The optimum growth temperatures were determined for the NZ,
CEU, Ku-2, and Ku-4 isolates.
These were consistant with the
temperature optima of the hosts from which the isolates were
obtained.
6.
Serological analysis demonstrated unique thermostable (0)
antigens for the NZ, CHU, Ku-2, and Ku-4 isolates.
72
7.
A common minor antigen was shared by two of the isolates
(Ku-i and Ku-5) from Kuwait.
Both isolates were recovered
from tilapia at the same location and had similar biochemical
reactions.
8.
Intraperitoneal infection of fingerling rainbow trout with
the NZ, CHU, Ku-2, and Ku-4 isolates produced a characteristic Gram-negative hemorrhagic septicemia.
9.
Two isolates recovered from tilapia had greater virulence for
rainbow trout than the two isolates recovered from saimonid
fish.
10.
Pathological signs observed in juvenile chinook salmon
following waterborne challenge with the two Kuwait isolates
were similar to the signs observed in fish challenged by
injection; however, the signs of the disease induced by
waterborne challenge progressed more slowly.
73
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