General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.

Users may download and print one copy of any publication from the public portal for the purpose of private study or research.

You may not further distribute the material or use it for any profit-making activity or commercial gain

You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from orbit.dtu.dk on: Feb 07, 2022

Identification and typing of Vibrio anguillarum: A comparison of different methods.

Austin, B.; Alsina, M.; Austin, D. A.; Blanch, A. R.; Grimont, F.; Grimont, P. A. D.; Jofre, J.; Koblavi, S.;Larsen, J. L.; Pedersen, KarlTotal number of authors:13

Published in:Systematic and Applied Microbiology

Link to article, DOI:10.1016/S0723-2020(11)80400-5

Publication date:1995

Document VersionPublisher's PDF, also known as Version of record

Link back to DTU Orbit

Citation (APA):Austin, B., Alsina, M., Austin, D. A., Blanch, A. R., Grimont, F., Grimont, P. A. D., Jofre, J., Koblavi, S., Larsen, J.L., Pedersen, K., Tiainen, T., Verdonck, L., & Swings, J. (1995). Identification and typing of Vibrio anguillarum: Acomparison of different methods. Systematic and Applied Microbiology, 18(2), 285-302.https://doi.org/10.1016/S0723-2020(11)80400-5

System. App!. !--.1icrobio!' 18,285-302 (1995) © Gustav Fischer Verlag, Stuttgart· Jena . New York

Identification and Typing of Vibrio anguillarum: A Comparison of Different Methods

B. AUSTIN" M. ALSINAb, D. A. AUSTIN', A. R. BLANCHh, F. GRIMONT", P. A. D. GRIMONTc, J. JOFREb ,

S. KOBLAVIc, J. L. LARSENd

, K. PEDERSENd, T. TIAINENd

, L. VERDONCKc, and J. SWINGS"

From the Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EHI4 4AS, Scotland'; Departament de Microbiologia, Universitat de Barcelona. Av. Diagonal 645, 08028 Barcelona, Spain"; Centre National de Typage Moleculaire Enterique, Unite des Enterobacteries, Institut Pasteur, 28 rue du Docteur Roux. F-75724 Paris Cedex \5, France\ Department of Veterinary Microbiolo~, Laboratory of Fish Diseases, Royal Veterinary and Agricultural University, Bulowsvej 13, DK-1870 Fre­deriksberg C, Denmark ; and Laborarorium voor Microbiologie, Faculteit Wetenschappen, Universiteit Gem, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium<

Received February 26, 1995

Summary

The majority (91 %) of 260 isolates initially identified as Vibrio anguillarum, that were obtained from a wide range of hosts, habitats and geographical locations, were recovered in a single cluster based on the ribotype and were pathogenic to Adamic salmon. A significant proportion of isolates (78% of the total) were allocated to 15 serogroups (01 - 010 and five previously undescribed groups referred ro as VaNTl, VaNT2, VaNT4, NaNT5 and VaNT7). A minority of isolates (6%) reacted with more than one antiserum or were self-agglutinating, and the remainder did not react with any of the antisera tested. Good correla­tion was noted between serogroups and lipopolysaccharide profiles, particularly with respect ro isolates belonging to serogroups 01, 02 and 04 - 010. Plasmids were recognized in some serogroups, especially 01, which contained the 67 kb plasmid associated with virulence. However, the) 9 profiles based on outer membrane protein patterns did not correspond to the results obtained with the other typing methods. Generally, the isolates were heterogeneous in their biochemical characteristics; 117 profiles were obtained with the API 20E system, and 9 and 32 clusters recognised from the results of BIOLOG fingerprinting and Biotype-l00 biotyping methods, respectively. Three dominant clusters were defined from fatty acid methyl esters profiles.

Key words: Vibrio - Fish isolates - Fish pathogens - Identification - Typing methods

Introduction

Vibriosis, caused by Vibrio anguillarum, is a significant limiting factor in mariculture, worldwide (Austin and Au­stin, 1993). Although formally reclassified into a newly proposed genus Listonella, as Listonella a1tguillara (Mac­Dcmell et aI., 1986), this classification was and is not widely accepted. Consequently, the pathogen is still com­monly referred to as V. anguillarum_ There is considerable heterogeneity between descriptions of the species (e_g_ West et aI., 1983; Kaper et aI., 1983; Larsen and Olsen, 1991), especially with regard to the recognition of a plethora of biotypes (Austin and Austin, 1993) and seroty­pes (Sorensen and Larsen, 1986). Initially, two biotypes, A and B (Nybelin, 1935) were recognised, followed by bioty-

pe C (Smith, 1961) and then biotype D and E (see Austin and Austin, 1993). Heterogeneity was recognised, insofar as two to four subgroups were defined after numerous taxonomy studies, including those of Kusuda et al. (1979), West et al. (1983) and Kaper et al. (1983). Furthermore, 10 serotypes/serogroups were defined (Muroga et aI., 1984; Kitao et aI., 1983; Sorensen and Larsen, 1986), some of which, e.g. serogroup 02, traversed species boundaries, i.e. V. anguillarum and V. ordalii (Larsen and Olsen, 1991). In part, this heterogeneity is a reflection of the ad hoc way in which vibriosis is often diagnosed, i.e. purely from pathological signs on diseased animals. Thus, the aetiological agent is frequently allocated to V. anguil-

286 B. Austin et al.

larum on no sound bacteriological basis (Allstin and AII­stin, 1993). When employed, identification techniques for pure cultures have relied heavily on serology, and biochemistry (Austin and Allstin, 1993). Attempts have been made to use rapid methods, such as the API 20E system (e.g. Kent, 1982; Maugeri et aI., 1983; Grisez et aI., 1991). However, the biochemical characteristics of V. anguillarum need clarification, because there is some dis­pute about the profile of the taxon (Larsen and Olsen, 1991; Wiik et aI., 1991). Although a diverse range of techniques have been used for identification and typing of V. angl/illarum (Austin and Austill, 1993), the compara­tive merits of the methods have not been assessed. Conse­quently, this study has sought to compare the efficacy of different methods for the identification and typing of cul­tures received as V. anguillarum.

Materials and Methods

Examination of cllitt/res. Two hundred and sixty cultures pro­visionally idenrified as V. angllillanmz. obtained from 17 species of fish, Artemia, rotifers, sediment and water in 16 countries, were examined (Table I). These were maintained in 20-25% (wi v) glycerol at -70, -80 or -40°c, and as plate cultures at room temperature on marine 2216E agar (Difco, Detroit, Michigan, USA) and tryptone soya agar (TSA; Oxoid, Basingsroke, Eng­land) supplemenred with 1 % or 1.5% (w/v) sodium chloride.

Ribotyping. Bacteria were grown in a shaking incubator for 18 h at 37°C in trypro casein soy (TCS) broth (Diagnostic Pasteur, Sanofi, France) supplemented with 2% (w/v) sodium chloride (TCSN). DNA was extracted from pellcts with the automate AutoGen 540 (AutoGen Instrumenrs, USA), and, after dialysis, cleaved for >3 h at 37°C with Bgn (Boehringer, Mannheim, Germany) (Grimont et aI., 1989). Restriction fragments were separated e1ectrophoretically through 0.8°;', (w/v) agarose (Ap­pligene, IIIkirch, France) in Tris-borate buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA buffer, pH 8.3) for 16 h at 1.5 VI cm, before transfer to nylon membranes (Hybond-N; Amersham International, Amersham, England) using a VacuGene System (Pharmacia). The DNA was fixed (15 min at 80°C) before hyb­ridization with an acetylaminofluorene (AAF)-labelled ribosomal probe (Eurogenrec, Seraing, Belgium), as described by Grimont et al. (1989). After blocking, the membranes were incubated at room temperature for 2-3 h with mouse anti-acetylaminofluor­ene monoclonal antibodies, and washed three times for 10 min in buffer (0.1 M maleic acid, 0.15 M NaCI, 0.3% Tween 20; pH 7.5) to eliminate excess antibody. Antimouse IgG alkaline phos­phatase-conjugated anribody (1 Ilg/ml) was added for 1 h, then, the systems were washed three times for 10 min. Revelation was in 0.1 M Tris HCI, 0.1 M NaCI, 50 mM MgCI 2 ; pH 9.5, after addition of nitroblue tetrazolium (Sigma) and 5 -bromo-4-chloro-3-indolyl phosphate (Sigma) as substrate. Purplish-blue bands appeared within a time span of a few minutes to one hour. The data were examined using the Dice (SAB) coefficient with cluster­ing by single linkage (Sneath and Sokal, 1973; Priest and Austin, 1993).

Serotyping. The preparation of absorbed pol}'donal antisera and O-antigen have been described previously (Maniatis et aI., 1982; Larsen et aI., 1994). A loopful of undiluted antiserum was mixed with an equal volume of O-antigen on a glass microscope slide, and agglutination reactions were recorded after visual in­spection.

Determination of lipopolysaccharide (LPS) profiles. LPS was extracted after enzymic digestion of lysed cells with proteinase K (Hitchcock and Brown, 1983), polyacrylamide electrophoresis using Mini-Protean" Ready Gels (Bio-Rad, J-krcules, California, USA) without 5DS, and with LPS silver stain (I\.tttelberger and Hilbink,1993).

Determination of outer membrane protein (OMP) profiles. OMP was extracted from young cells (overnight broth cultures) which were disrupted by alternate freezing and thawing in Tris­HCI (10 mM) buffer prepared in 0.3% (w/v) sodium chloride with the sarkosyl technique (tambert. 1986), polyacrylamide electrophoresis using Mini-Protean" Ready Gels (Bio-Rad) with­out 5DS, and later with Coomassie Brillianr Blue stain. The gels were stained with protein-silver stain (Merril et aI., 1981), as necessary.

Gas chr01l1atogr,lphic analysis of cellular fatty acid methyl es­ters (FAMEs). A subset of 147 V. anguillartlm cultures was ex­amined by this method. Quanritative analysis of cellular fatty acid compositions was performed using the gas-liquid chromato­graphic procedure, as described by De Boer and Sasser (1986). Cultures were grown for 24 h at 28°C on TSA supplemented with 1.5% (w/v) sodium chloride. Approximately 70 mg of cells were added to 1 ml of 3.75 M NaOH in 50% (v/v) aqueous methanol, and heated for 30 min in a boiling water bath for saponification. Methylation was achieved by adding 2 ml of 6 N hydrochloric acid in 50% (v/v) aqueous methanol and heating for 10 min at 80°C. After cooling to room temperature, fatty acid methyl esters (FAMEs) were extracted with a 1: I mixture of hexane and methyl-iso butylether. The FAMEs were analysed with a Hewlett-Packard model 5898A gas chromatograph and identified using the Microbial Identification System software pac­kage (MIS version no. 3.7, obtained from Microbial ID Inc., Newark, Delaware, USA). Clustering was by numerical analysis using the Euclidean distance coefficient and the unweighted pair­group method of averages (UPGMA; Sneath and Sakal, 1973; Priest and Austin, 1993). Clusters were delineated at Euclidean distance 69.

Plasmid profiling. Overnight cultures in Luria-Berrani broth base (LB; Gibco, Paisley, Scotland) or veal infusion broth (Difco) supplemented with 0.5% (w/v) sodium chloride were used for the determination of plasmid profiles. The plasmids were extracted from the bacteria by the method of Kado and Liu (1981). Plas­mids were separated by gel electrophoresis in 0.6-1.0% agarose gels (SeaKem GTG; FMC, Bio Products, USA) in T AE buffer, at pH 8.0. The size of the plasmids was determined by the length of migration, according to the method of Rochelle et al. (1985). All cultures were examined at least four rimes.

Examination of ClIltllres by the API 20E rapid idmtifiwtion s)'stem. Inocula were prepared from 24-28 h cultures incubated at 20 DC on marine 2216E agar (Difco). Cells were harvested in phosphate buffered saline (PBS) supplemenred with 2% (w/v) sodium chloride, and the suspensions adjusted with fresh PBS to achieve a standard opacity equivalent to McFarland No.3. The API 20E rapid identification sysrems (BioMerieux, Marcy­l'Etoile, France) were inoculated using the modification for marine bacteria described by Kent (1982) and Grisez er al. (1991) with incubation at 20°C for 24 and 48 h.

BIOLOG fingerprints. Strains were grown for 24 h at 25 DC on brain heart infusion agar (Difco) supplemented with 1.5% (wI v) sodium chloride. Inocula were prepared in 1.5% (w/v) sodium chloride, and the cell density standardized (0 berween 0.261 and 0.3 OD using a spectrophotometer at 590 nm. Each well in the BIOLOG GN microplate (Biolog Inc., Hayward, CA., USA) was inoculated with 150 fd of cell suspension, and the microplates were incubated at 25°C for 24 h. Changes in colour were mea­sured using a Multiscan Multisoft filter photometer (Labsystems, Helsinki, Finland) at 590 nm. The BIOLOG profiles were com-

pared by numerical analysis (PhenoCompar, L. Vallterin) with similarities calculated by the Pearson product moment correla­tion coefficient between strains_ The strains were grouped by UPGMA. Phenotypic clusters were delineated at 80% r. Differen­tiating characters between pairs of clusters were calculated using the BIONUM software package (L. Vallterin et al.), and were defined as those tests yielding >90% and < 10% of positive re­sponses, respectively. In order to extract differentiating charac­ters, optical density values were recalculated between maximum and minimum values, whereby the cut-off factor to obtain positives and negatives was set at 30%. The internal homogeneity within each delineated cluster was calculated using the same soft­ware package.

Biotyping. Carbon source utilization tests were carried out using Biotype-IOO strips (BioMerieux; La Balme-Ies Grones, France) that contained 99 pure carbon sources. Volumes (60 ml) of Biotype medium No.1 were supplemented with 2% (w/v) of sodium chloride and 100 mM MgClb and inoculated with 2 ml amounts of a 100- Klett unit suspension of bacterial cells in 2 % (w/v) sodium chloride. The strips were completely filled so as ro obtain a slightly convex meniscus, and incubated at 30°C for 2 and 4 days. The cupules were examined visually for the presence of growth, and the data were examined by the Jaccard (S)) coeffi­cient with UPGMA clustering (Sneath and Sokal, 1973; Priest and Austin, 1993), using the Recognizer program (Institut Pas­teur Taxolab, Paris, France).

Fish pathogenicity experiments. Bacterial cultures were grown overnight at 22°C in tryptone soya broth (Oxoid) supplemented with 1 % (w/v) sodium chloride, centrifuged at 5,000 x 'g' for 10 min and resuspended in 10 ml volumes of 0.9% (w/v) saline to approximately ION cellslml. Ten fold dilutions were prepared to 104 cells/ml. A salmonid fish model (Atlantic salmon [Salma sa­lar]) was used to assess pathogenicity, as follows:

Groups of 10 fish wre infected by intraperitoneal injection with 0.1 ml volumes of the washed bacterial suspensions to achieve doses of 104 _106 cells/fish. The infected animals were maintained for up to 14 days in covered polypropylene tanks supplied with dechlorinated, aerated static freshwater (the water was changed daily) at a temperature of -15°C. Dead and moribund fish were removed, and subjected to standard bac­teriological and pathological examination (Austin and Allstin, 1989). Any survivors at the end of the experiment were sacrificed and examined, as above. The disease signs were recorded, and attempts made to recover and confirm the identity of the patho­gen by the methods described by Allstin and Austin (1993).

Test reproducibility. The test results for 20 duplicate cultures were compared for all methods, and an estimate of individual test variance (S,2) was calculated (formula 15; Sneath and Johnson. 1972). The average test variance was calculated to obtain a pooled variance (52), which was used to estimate the average probability (p) of an erroneous test result (formula 4; Sneath and Johnsorl, 1972).

Results

1. Test reproducibility

Inclusion of duplicate isolates in the study enabled ex­perimental test error to be estimated. The average proba­bility (p) of an erroneous result was 5_6%, calculated from the pooled variance (52 = 0.0558) of all the methods scored for the duplicate cultures. Values of 5,z ranged from zero for some of the test regimes, e.g. ribotyping, through 0.03 for OMP and pathogenicity, 0.05 for serotyping, 0.15 for LPS profiles, 0.44 for the API 20E system, and 0_9 for BIOLOG.

Identification and Typing of Vibrio angllillarum 287

2. Ribotypillg

The majority (236/260 = 91 %) of the cultures were ascribed to a single ribotype cluster defined at or above the 80% similarity level (Table 1; Fig. 1). The cultures in this cluster were characterised by three groups of bands. Thus, there were 2-7,4-9 and 2 bands between 10 kb and 20 kb, 4 kb and 8 kb, and 2 kb, respectively. The remaining 24 cultures were recovered in 19 separate clusters. Of these, clusters 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15-20 comprised single isolates; clusters 5, 13 and 14 each con­tained two isolates, and cluster 7 comprised 6 isolates (Table 1; Fig. 1).

% SIMILARITY 40 !

100 rlU~rn ~O Of mm ij~ oour~ IDENTITY

mmrt

2

3 4 5 6

7

8 9

\0 11

12 \3 14

IS 16

17 18 11)

20

236

1 2

6

2 2

v. anguillarum

VIB 225

VIB66

VIB 76 VIB 189. VIB 190 VlB 239

VIB 192. VIB 193. VIB 195. VIB 210. VIB 21I, VIB 223 VIB 224 VlB 212

VIB 194 VlB 215

VIB 23 VlB 186, VIB 213 VlB 144. VIB 145

VlB 187 VlB 218 VIB 191 VIB 214

VIB 185 VlB IR4

Fig. 1. Simplified dendrogram based on the examination of ribo­typing data by the SAS coefficient and single linkage clustering.

Tab

le 1

. B

acte

rial

cul

ture

s tv

0

0

00

Lab

orat

ory

Col

lect

ion'

/ So

urce

and

/or

coun

try

Rib

otyp

e S

ero·

LP

S O

MP

FA

:VIE

Pl

asm

id

BIO

LO

G

API

20E

B

ioty

pe

Pat

ho-

refe

renc

e no

. st

rain

no.

of

ori

gin

clus

ter

grou

p pr

ofil

e pr

ofil

e pr

ofil

e c

on

terl

( pr

ofil

e pr

ofil

e pr

ofil

e ge

nici

ty

?'

VIB

1

AT

CC

433

05

Ollc

orh)

'IIc/

ms

lIIy

kiss

, 0

1

2 2

67 k

b 30

4752

556

+

>- c D

enm

ark

~

VIB

2

AT

CC

433

06

Gad

us m

orim

a, D

enm

ark

02

a

2 1

2 0

3247

5275

7 +

5'

n

VIB

3

AT

CC

433

07

O. m

ykis

s, D

enm

ark

03

3

3 2

0 32

4752

757

25

+

~ '"

VIB

4

AT

CC

433

08

G.l

llor

hua,

Den

mar

k 0

4

4 2

2 0

3247

5275

7 15

+

:-

VIB

5

AT

CC

433

09

G. m

orhu

a, D

enm

ark

05

5

3 2

0 30

4712

557

30

+

VIB

6

AT

CC

43

3\0

G

.lll

orhu

a, D

enm

ark

06

6

3 2

0 30

4752

757

+

VIB

7

AT

CC

433

11

Ang

uill

a an

guill

a, D

enm

ark

07

7

1 2

0 32

4752

757

15

+

VIB

8

AT

CC

433

12

G. m

orl"

,a,

Den

mar

k 0

8

8 1

2 0

3247

5255

7 16

+

V

IB 9

A

TC

C 4

3313

G

. mor

hua,

Den

mar

k 0

9

9 3

2 0

1247

1255

7 +

V

IB 1

0 A

TC

C 4

3314

C

. mor

hua,

Den

mar

k 10

10

.3

2 0

3247

5275

7 27

+

(w

eak)

V

IB 1

1 A

VL

90-9

-22

Dic

entr

arch

us la

bm.>

:, 0

1

1 3

67 k

b 30

4773

757

23

+

Gre

ece

V1B

12

AV

L 89

-2-5

4 D

.lab

rax,

Gre

ece

02

3

2 2

2 0

3047

5164

7 24

+

V

IB 1

3 A

VL

68-2

8-C

D

.lab

rax,

Gre

ece

02

b

2 1

0 32

4753

757

8 +

V

IB 1

4 H

WU

44

O. m

ykis

s, I

taly

0

1

3 67

kb

3247

7375

7 23

+

V

IB 1

5 H

WU

48

D.la

hra.

>:,

Gre

ece

01

3

67 k

b 32

4752

457

I +

V

IB 1

6 H

WU

49

D.l

ahra

x, G

reec

e 0

1

2 6

7k

b

3047

7245

7 4

+

VIB

17

HW

U 5

0 D

. lah

rax,

Gre

ece

01

3

67 k

b 30

4753

757

2 +

V

IB 1

8 A

VL

27.

2 O

. myk

iss,

Den

mar

k 0

1

2 2

50

,67

kb

3046

7375

7 +

V

IB 1

9 A

VL

2&.2

O

.III

),ki

ss,

Den

mar

k 0

1

1 4

50

,67

kb

I 30

4773

757

+

VIB

20

AV

L 2

8.3

O. m

),ki

ss,

Den

mar

k 1

01

I

2 5

0,6

7 k

b I

3247

7375

7 32

+

V

IB 2

1 A

VL

28.

4 O

. m),

kiss

, D

enm

ark

I 0

1

1 2

50

,67

kb

I 72

4753

757

+

VIB

22

AV

L 20

/90

D.l

abra

x, G

reec

e I

05

24

10

4

6,7

6 k

b 8

5346

2255

7 +

V

IB 2

3 A

VL

33/

91

Spar

us t

lura

ta,

Gre

ece

12

05

24

10

76

kb

8 53

4622

557

+

VIB

24

AV

L 1

8/91

Sp

. aur

ata,

Gre

ece

24

10

1 5

0,7

0 k

b 8

5346

2255

7 +

V

IB 2

5 H

WU

63

Sa

llllo

sal

ar,

Chi

le

01

1

2 3

67 k

b 1

3047

7275

7 +

V

IB 2

6 H

WU

64

fish

02

b

2 0

1 32

4712

757

9 +

V

IB 2

8 H

WU

V0

2

Sa.

sala

r, N

orw

ay

01

2

2 67

kb

3047

5275

7 +

V

IB 2

9 H

WU

VA

73

D.l

ahra

x, G

reec

e cr

oss-

2 2

0 32

4712

657

+

reac

tion

s be

twee

n 0

2a&

05

V

IB 3

0 H

WU

VA

75

Sp. a

ura

ta,

Gre

ece

(ros

s-2

3 0

3247

1265

7 7

+

reac

tion

s be

twee

n 0

2a&

05

V

IB 3

1 N

CM

B 8

28

Sa. s

afar

, U

.K.

02

a

2 2

2 0

3247

5245

7 +

V

IB 3

2 N

CM

B 5

72

O. m

ykis

s. J

apan

0

1

2 67

kb

3047

5275

7 +

V

IB 3

4 U

B 0

1/91

~Iugilidac,

Ital

y 0

1

1 3

67 k

b 32

4752

457

2 +

" V

IB 3

5 U

B 2

8/89

D

.lab

rax,

Ira

ly

01

1

2 67

kb

3247

5245

7 2

+

VIB

36

UB

76/

91

Mug

ilid

ae,

Ital

y 0

1

1 2

2 67

kb

3247

5245

7 +

(w

eak)

V

IB 3

7 U

B 1

09/9

0 Sp

. aI/

rata

, It

aly

04

4

19

0 32

4752

757

25

+

VIB

39

UB

178/

90

D.l

ahra

x, I

tal)

, 0

1

1 1

2 67

kb

3247

5244

7 +

V

IB 4

0 U

B 19

1/90

D

.lab

rax,

Ita

l),

01

1

78,

84 k

b 32

4752

557

+

VIB

41

UB

2071

90

Sp. a

urat

a, I

taly

08

8 0

3247

1245

7 +

Tab

le I

. C

on

tin

ued

Lab

orat

ory

Col

lect

ion'

1 S

ourc

e an

dlor

cou

ntry

R

ibot

)'pe

Sero

-LP

S O

MP

F

AM

E

Pla

smid

B

IOL

OG

A

PI2

0E

B

iot)

'pe

Pat

ho-

refe

renc

e no

. sr

rain

no.

of

ori

gin

clus

rer

grou

p pr

ofil

e pr

ofil

e pr

ofil

e co

nten

t pr

ofil

e pr

ofil

e pr

ofil

e ge

nici

t)'

VlB

42

UB

217

/90

D./

abra

x, I

taly

2

2 0

3247

5275

7 +

V

IB 4

4 U

B 2

6119

1 D

./ab

rax,

Ita

ly

0)

1 1

35

,60

kb

3247

5275

7 +

VIB

45

UB

295

/91

D./

abra

x, I

taly

0

4

4 12

0

3247

5257

7 +

" V

IB 4

6 U

B 3

27/9

0 D

./ab

rax,

Ita

ly

01

I

2 67

kb

3247

5245

7 1

,3

+"

VlB

47

UB

342

191

D./

abra

x, I

taly

0

1

1 2

67 b

32

4752

457

1 +

b V

lB 4

8 U

B 3

9319

1 D

./ab

rax,

Ira

l)'

01

1

2 67

, 84

kb

3247

5265

7 23

+

" V

lB4

9

UB

397

/90

D.l

abra

x, I

taly

4

2

3 0

3247

5276

7 +"

V

lB 5

0 U

B 4

17/9

0 D

./ab

rax,

Ita

ly

02

a

2 2

0 32

4752

446

+

VIB

5!

UB

498

/90

fi.h,

Ita

ly

02

a

42

3 0

3247

5275

7 15

+

V

IB 5

2

UB

505

/91

D./

abra

x, I

taly

46

1

2 0

3247

5257

7 16

+

V

IB 5

3 U

B 2

58/9

1 D

./ab

rax,

Ita

ly

02

a

2 2

0 32

4732

657

+ V

IB 5

4 U

B 5

74/9

1 Sp

. aur

ata,

Ita

ly

07

7

2 0

3247

5255

7 +

V

IS 5

5 U

B 5

78/9

0 D

. /a

brax

, It

aly

02

a

2 2

0 32

4732

657

6 +

V

lB 5

6 U

B 6

01

19

) D

./ab

rax,

Ita

ly

01

2

67

,84

kb

-32

4752

657

+

VlB

57

UB

86

1/8

9

D./

abra

x, I

raly

0

1

1 2

67 k

b 32

4752

757

+

VIB

58

UB

904

/89

D./

abra

x, I

taly

13

6

0 32

4752

757

15

+ V

IB 5

9 U

S 90

9/89

D

.lab

rax,

Ita

ly

01

1

1 0

3247

5245

7 +

(w

eak)

V

IB 6

0 U

B 9

10/9

0 Sp

. aur

ata,

Ita

ly

03

42

13

0

3247

5277

7 31

+

V

IB 6

1 U

B 9

60/9

0 D

.lab

rax,

Ita

ly

03

42

2

2 0

3247

5265

7 27

+

" V

IB 6

2 U

B 1

0321

90

D./

abra

x, I

taly

0

3

42

2 0

3047

5275

7 V

IB 6

3 U

B 1

075/

90

D./

abra

x, I

taly

0

3

42

2 0

7347

5275

7 24

+

V

IB 6

4 U

B A

023

Scop

htha

/mus

max

im/I

S,

01

2

2 26

kb

3047

5275

7 23

+

Spai

n V

IB 6

5 U

B A

024

Sc. m

axim

us,

Spa

in

1 0

1

1 2

67 k

b

3247

5275

7 +

c;:

V

IB 6

6 U

B A

044

Sc. m

axim

/IS,

Spa

in

3 8

11

0 30

4652

556

+

" V

IB 6

7 U

B A

055

Sc. m

axim

us.

Spa

in

01

1

2 67

kb

3247

7265

7 23

+

" a

VlB

68

UB

A05

6 Sc

. max

imu5

, S

pain

0

1

1 2

67 k

b 30

4652

457

23

+

n'

~

VIB

69

UB

A07

S Sp

. allr

ata,

Spa

in

02a

2 6

2 0

3247

5275

7 24

+

o·

VIB

70

UB

A08

5 ro

tife

r, S

pain

5

2 0

3047

5275

7 15

+

" ::J

'" V

IB 7

1 L

MG

441

1 S

cotl

and

02

a

14

3 2

0 4

3247

5245

6 " c...

VlB

72

L

MG

443

7T

G. m

orhu

a, N

orw

ay

02

a

2 2

2 0

1 32

4752

757

+

-I

VIB

73

LM

G 1

1684

1

02

a

2 2

2 0

3247

5264

7 .....

+

""

VlB

75

LM

G 1

0939

U

.S.A

. 1

01

I

2 2

67

kb

4

3207

5275

6 +

5 C/O

VIB

76

LM

G 1

2098

Ja

pan

4 0

2a

2 1

2 0

4 30

4752

547

+

~

VIB

77

LM

G 1

2099

P/

ecog

loss

/4S

a/tiv

e/is

. Ja

pan

I 0

2a,

b

2 6

2 0

3247

5255

7 19

+

~

VIS

78

LM

G 1

2100

P.

a/ti

ve/is

, Jap

an

1 14

9

2 5

0 k

b

3047

1245

7 +

<3

-

VIS

79

LM

GI2

1O

! 0

1

1 2

2 77

kb

3047

5265

7 +

a<

VIS

80

LM

G 1

2102

0

2a

2 9

2 0

3047

3265

7 +

t:.

VIB

81

LM

G 1

3185

D

./ab

rax,

Gre

ece

cros

s 43

1

2 4

6,6

8,

3247

7255

7 16

+

~ ::

reac

tion

s 83

kb

~ V

IS 8

2 L

MG

131

86

D.l

abra

x. G

reec

e V

aNT

2 43

1

2 6

8,8

3 k

b 32

4772

577

10

+ ...

VIB

83

LM

G 1

3187

D

./ab

rax,

Fra

nce

01

1

2 2

0 30

4752

457

1 +

(w

eak)

~

VIS

84

LM

G 1

3188

D

./ab

rax,

Ita

ly

01

2

2 67

kb

3047

7245

7 +

VlB

85

RV

AU

87-

9-11

7 O

. myk

iss,

Fin

land

0

1

2 67

kb

3247

7265

7 +

b

VIB

86

RV

AU

169

2 O

. myk

iss,

Ita

ly

01

2

67 k

b 30

4752

757

+

1'-'

00

\0

Tah

le I

. C

on

tin

ued

, .... 'C

0

Lab

orat

ory

Col

lect

ion"

1 S

ourc

e an

d/or

cou

ntry

R

ibot

ype

Ser

o·

LPS

OM

P

FA!v

IE

Pla

smid

B

IOL

OG

A

PI2

0E

B

ioty

pe

Pat

ho·

refe

renc

e no

. st

rain

no.

o

f or

igin

cl

uste

r gr

oup

prof

ile

prof

ile

prof

ile

cont

ent

prof

ile

prof

ile

prof

ile

geni

city

?"

V

IB 8

7 N

CM

B 1

873

Sa. s

al"r

, U

.K.

cros

s 2

5.3,

11.

5,

30

47

72

65

7

+ (w

eak)

;.

.

reac

tion

s 5

3k

b

~ be

twee

n :5' "

01

&

M '"

VaN

Tl

-V

IB 8

8 R

VA

U 5

1182

12

O. m

ykis

s, G

erm

any

01

1

2 67

, 10

5 kb

3

04

77

26

57

V

IB 8

9 R

VA

U 8

2061

7·11

6 O

. myk

iss,

Den

mar

k 0

1

1 2

67

kb

3247

5275

7 +

VIB

90

RV

AU

820

901·

2/2

O. m

ykis

s, S

wed

en

01

I

2 67

kb

3247

5274

7 +

h V

IB 9

1 R

VA

U 8

3042

2·1/

1 O

. myk

iss,

Den

mar

k 0

1

I 2

2 67

kb

3247

7275

7 +

VIB

92

RV

AU

810

530·

111

O. m

ykis

s, D

enm

ark

01

1

2 67

kb

1 32

4772

757

+ V

IB 9

3 R

VA

U 8

5061

O-1

/6a

O. m

ykis

s, D

enm

ark

01

I

2 67

kb

I 32

4772

757

VIB

94

NC

MB

829

O

. myk

iss,

U.K

. 0

2a

2 4

2 0

4 32

4752

456

8 +

VIB

95

RV

AU

222

8/80

sa

lmon

, S

wed

en

02

a

2 2

0 32

4750

747

+ V

IB 9

6 R

VA

U 2

887

O. m

yki5

s, I

taly

0

2a

2 2

2 64

, 32

4732

657

+ -2

00

kh

VIB

97

RV

AU

850

617·

1/1

Eso

x lu

cius

, F

inla

nd

02

a

2 I

0 32

4752

757

18

+ V

IB 9

8 R

VA

U 8

6090

8·3

Sc. m

axim

us,

Nor

way

0

2a

2 2

0 30

4752

657

21

+ (w

eak)

V

IB 9

9 R

VA

U 8

9-2-

62

Sc. m

axim

lls,

Nor

way

0

2a

2 7

2 0

3047

5375

7 +

VIB

100

R

VA

U 9

1-7-

175

Sc. m

axim

lls,

Den

mar

k 0

2a

2 2

0 32

4712

757

7 +

(wea

k)

VIB

101

R

VA

U 8

2072

3-21

8 Sc

. max

imus

, S

wed

en

02

b

2 I

2 2

0 k

b 30

4752

757

7 +

(wea

k)

VIB

102

R

VA

U 1

076/

2 G

. mor

hlla

, D

enm

ark

02

b

2 2

0 30

4752

457

14

+ V

IB 1

03

RV

AU

108

9/2

G. m

orhu

a, D

enm

ark

02

b

2 1

>2

00

kb

3247

5245

6 14

+

VIB

104

R

VA

U 1

4741

1 G

. mor

hua,

Den

mar

k 0

2b

2

7 2

>2

00

kb

3247

5275

7 +

VIB

105

R

VA

U 6

828c

Sc

. max

illlu

s, D

enm

ark

02

b

2 7

0 32

4752

757

14

+ V

IB 1

06

RV

AU

25/

82/2

O

.lIIy

kiss

, G

erm

an),

0

2b

2

2 0

3247

5275

7 +

VIB

108

R

VA

U 8

6061

9·3/

2 O

. lII

ykis

s, D

enm

ark

02

b

2 2

2 >

20

0 k

b 32

4772

757

+ V

IB 1

09

RV

AU

860

828-

1I3a

O

. myk

iss,

Den

mar

k 0

2b

2

2 0

3247

7275

7 +b

V

IB 1

10

RV

AU

90·

2-6

Pol

lach

il/s

(lire

llS,

Nor

way

0

2b

2

2 2

>2

00

kb

3247

3245

6 +

(wea

k)

VIB

III

R

VA

U 1

3211

1 G

. m

orhu

a, D

enm

ark

self

agg

l-45

2

2 0

3247

5265

7 +b

ur

inat

ing

VIB

112

R

VA

U 8

2080

6·11

2 O

. myk

iss,

Den

mar

k cr

oss

45

3 0

3247

5275

7 28

re

acti

ons

VIB

113

R

VA

U 8

2080

6-1/

3 O

.lIIy

kiss

, D

enm

ark

03

3

.3 2

0 3

24

75

27

57

28

V

IB 1

14

RV

AU

171

2/1

G. m

orhl

/a,

Den

mar

k 0

4

4 2

2 0

3247

5275

7 2

0

VIS

115

R

VA

U 7

4/82

/1

O. m

ykis

s, G

erm

any

04

4

8 0

3247

5275

7 V

IB 1

17

RV

AU

89·

9·18

1 Sa

. sal

ar,

Nor

way

0

4

4 8

2 0

32

47

52

75

7

VIB

118

R

VA

U 1

629

G. m

orhl

/a,

Den

mar

k 0

5

5 I

2 0

30

47

12

55

7

VIB

119

R

VA

U 8

5081

6-11

17b

O. m

ykis

5, D

enm

ark

05

5

1 2

0 32

4712

557

VIB

120

R

VA

U 9

0-2-

9 D

.lab

rax,

Nor

way

15

2

0 3

04

75

27

57

15

+

V

IB 1

22

RV

AU

682

8g

Sc. m

axill

lus,

Den

mar

k 0

7

7 3

0 3

24

75

27

57

+h

V

IB 1

23

RV

AU

170

113b

A

ngui

lla s

p.,

Den

mar

k cr

oss

2 14

0

3247

5245

6 8

+ re

acti

ons

VIB

124

R

VA

U 1

7021

1 A

ngui

lla s

p.,

Den

mar

k 0

7

7 3

0 3

04

75

27

57

15

+b

V

IB 1

25

RV

AU

88-

12-7

16

Sa.

sala

r, N

orw

ay

08

8

2 0

3247

5257

7 16

+

VIB

126

R

VA

U 8

4060

6·21

5 O

. myk

iss,

Den

mar

k V

aNT

I 45

2

53

,56

kb

3247

5275

7 23

+b

V

IB 1

27

RVAU

373

7/1

O. m

ykiss

, Ita

ly

VaN

T4

47

28

2 0

3247

3245

7 +

,~

Tab

le I

. C

onti

nued

0 ~

< ~

Lab

orat

or),

C

olle

ctio

n'l

Sour

ce a

nd/o

r co

untr

),

Rib

otyp

e Se

ro-

LPS

OM

P

FAM

E

Plas

mid

B

IOL

OG

A

PI 2

0E

Bio

t)'p

e Pa

th,,-

;,-re

fere

nce

no_

stra

in n

o.

of o

rigi

n du

ster

gr

oup

prof

ile

prof

ile

prof

ile

cont

ent

prof

ile

prof

ile

prof

ile

geni

cit)

' .., "2

- ~ V

IB 1

28

RV

AU

850

610-

lIS

b O

. myk

iss,

Den

mar

k V

aNT

2 0

3 2

11.5

kb

3247

5275

7 +

" g.

VIB

129

R

VA

U 8

5061

7-11

3 G

. mor

hua,

Fin

land

V

aNT

5 43

2

0 72

4752

557

+

~ V

IB 1

30

RV

AU

91-

7-15

2 Sc

. max

im us

, D

enm

ark

2 2

2 0

3246

1055

6

~ V

IB 1

31

RV

AU

88-

6-73

12

O.

myk

iss,

Den

mar

k 0

2a

2 2

2 5

,5.5

, 32

4752

757

+

11 k

b "" .~

VIB

132

R

VA

U 9

1-7-

143

0_ m

),ki

ss,

Den

mar

k 0

3

0 2

2 0

3247

5275

7 V

IB 1

34

RV

AU

91-

8-17

8 Sc

. max

im us

, N

orw

ay

01

2

53

,67

kb

3247

5275

7 23

+

V

IB 1

35

RV

AU

91-

11-2

44

Sc. m

axim

us,

Nor

way

0

1

I 2

2 3

3,6

7 k

b 32

4772

757

21

+"

VIS

136

R

VA

U 9

2-8-

158

All

guil

la s

p.,

Den

mar

k 0

3

3 2

0 32

4752

757

17

+

VIB

137

R

VA

U 9

2-8-

161

Allg

uilla

sp.

, D

enm

ark

02

a

2 2

0 30

4652

457

+

VlB

138

R

VA

U 9

2-8-

163

Allg

uilla

sp.

, D

enm

ark

1 0

2a

2 2

2 0

1046

5245

6 8

+

VIB

139

R

VA

U 9

2-8-

\68

Ang

uill

a sp

., D

enm

ark

I 0

3

3 2

2 0

3247

5275

7 +

V

IB 1

40

RV

AU

910

614-

111

G. m

orhu

a, D

enm

ark

I 0

2a

2 2

0 32

4712

657

5 +

V

IB 1

41

RV

AU

840

523-

2f4b

O

. myk

iss,

Den

mar

k I

VaN

T2

45

3 0

3247

7275

7 13

V

IB 1

42

R V

AU

840

808-

3/4a

O

. m),

kiss

, D

enm

ark

I V

aNT

2 2

3 2

0 7

7246

1255

7 V

IB 1

43

RV

AU

VI

312

wat

er,

Den

mar

k I

01

I

2 0

I 32

4752

577

VIB

144

R

VA

U V

i 4/

2 w

ater

. D

enm

ark

14

48

2 0

7 32

4612

547

VIB

145

R

VA

U V

I 5/

2 w

ater

, D

enm

ark

14

48

2 2

0 7

7046

1255

7 V

IB 1

46

RV

AU

V2

112

wat

er,

Den

mar

k 0

2a

21

2 0

7346

1265

7 +

V

IB 1

47

RV

AU

V2

2/2

wat

er,

Den

mar

k 0

9

0 2

7347

5245

7 V

IB 1

48

RV

AU

V2

4/3

wat

er,

Den

mar

k 0

3

45

I 2

0 1

3247

5275

7 +

(w

eak)

V

IB 1

49

RV

AU

V2

5/3

wat

er,

Den

mar

k V

aNT

2 45

3

2 0

3247

7275

7 12

V

IB 1

50

RV

AU

VI

1/4

wat

er.

Den

mar

k 0

2 2

0 32

4752

777

17

VIB

151

R

VA

U V

I 2/

4 w

ater

, D

enm

ark

0 2

2 0

3247

1275

7 c;:

V

IB 1

52

RV

AU

V2

114

wat

er,

Den

mar

k 0

3

3 2

0 32

4752

757

17

+

" g V

IB 1

53

RV

AU

Sel

112

se

dim

ent,

Den

mar

k 0

3 0

3247

5275

7 21

S1

V

IB 1

54

RV

AU

Sci

3/2

se

dim

ent,

Den

mar

k 0

5

I 0

3247

5275

7 22

,.,

+

~

VIB

ISS

R

VA

U S

e1 5

13

sedi

men

t, D

enm

ark

I 3

2 0

3247

5275

7 +

o·

sedi

men

t, D

enm

ark

05

2

2 2

0 32

4712

557

::>

VIB

156

R

VA

U S

e2 1

/2

., V

IB 1

57

RV

AU

Se2

2/2

se

dim

ent,

Den

mar

k 0

5

2 2

0 32

4712

557

30

~

VIB

158

R

VA

U S

e22/

4 se

dim

ent,

Den

mar

k 4

2 2

0 32

4772

757

...;

VIB

159

R

VA

U S

ci 3

/4

sedi

men

t, D

enm

ark

08

69

2

2 0

3257

0254

7 29

. .,

+

"0

VIB

160

R

VA

U S

el l

iS

sedi

men

t, D

enm

ark

02

a

2 2

2 0

3247

5255

7 5'

Q<

O

VIB

161

R

VA

U S

el 5

15

sedi

men

t, D

enm

ark

cros

s 44

0

3247

1255

7 29

0 .....

re

acti

ons

~ V

IB 1

62

RV

AU

Se2

315

sedi

men

t, D

enm

ark

04

4

18

0 32

4752

752

20

~.

VIS

163

R

VA

U P

T24

P.

a/ti

ve/is

, Ja

pan

01

1

2 0

3047

5275

7 +

(w

eak)

0

VIB

164

R

VA

U P

T49

3 P.

a/ti

velis

, Ja

pan

cros

s 45

35

2

0 32

4752

756

25

+

'" ;:s

reac

tion

s Q

\:)

;:

VIB

165

R

VA

U P

T21

3 P.

a/ti

velis

, Ja

pan

01

2

2 0

1 30

4752

757

~ V

IB 1

66

RV

AU

PB

I5

P. a

/tive

lis, J

apan

0

5

2 2.

6 kb

I

i1 V

IS 1

67

RV

AU

PB

28

P. a

/tive

/is,

Japa

n 45

1

2 0

4 30

0612

457

::! V

IB 1

68

RV

AU

ET

I P.

a/ti

ve/is

, Ja

pan

07

(w

eak)

0

I 30

4752

557

19

VIB

169

R

VA

U 7

75

salm

on.

U.S

.A.

01

2

2 67

kb

3207

7265

7 +

V

IB 1

70

RV

AU

53-

507

salm

on,

U.S

.A.

01

2

2 67

kb

3247

7275

7 17

+

1'

-'

RV

AU

RG

75-

834

salm

on,

U.S

.A.

01

2

2 6

7,3

6 k

b 32

4752

757

'Ci

VIB

171

+

Tab

le I

. C

on

tin

ued

I'

" -0

t"

Llb

ora

tory

C

olle

ctio

n'/

Sou

rce

and/

or c

ount

ry

Rib

otyp

e S

ero-

LPS

OM

P

fAM

E

Pla

smid

B

IOL

OG

A

PI2

0E

B

ioty

pe

Pat

ho-

refe

renc

e no

. st

rain

no.

of

ori

gin

clus

ter

grou

p pr

ofil

e pr

ofil

e pr

ofil

e co

nten

t pr

ofil

e pr

ofil

e pr

ofil

e gc

nici

t)'

?'

VIB

17

5 R

VA

U V

AI2

O

. myk

iss,

Can

ada

01

2

67 k

b 3

24

77

27

57

+

(w

eak)

;..

V

IB 1

76

RV

AU

VA

41

O. m

~'ki

ss,

Can

ada

01

2

2 6

7 k

b 3

24

77

27

57

+

" ~

VIB

17

8 R

VA

U V

A43

O

. Iny

kiss

, C

anad

a 0

1

2 67

kb

30

47

72

75

7

+

5'

VIB

18

0 R

VA

U V

A61

O

. ne

rka,

Can

ada

01

2

67 k

b 3

24

77

27

57

+

~

VIB

181

R

VA

U V

A70

SI

I. sa

/ar,

Can

ada

01

2

67 k

b 3

24

77

27

57

.,

+

:-V

IB 1

82

RV

AU

VA

72

O. m

ykis

s. C

anad

a 0

1

I 2

67 k

b 1

32

47

72

75

7

+

VIB

18

4 L

MG

132

11

Art

emia

, S

pain

29

19

5

2 0

3 3

04

41

37

57

+

V

IB 1

85

l.M

G 1

3212

A

rtem

ia.

Spdi

n 19

0

4

16

16

2 32

kb

8 4

14

71

25

56

+

V

IB 1

86

LM

G 1

3213

A

rtem

ia,

Spa

in

13

19

5 2

4.4

kb

.3

72

44

13

75

6

+

VIB

187

L

MG

132

14

D./

abra

x, S

pain

15

w

eak

17

3 2

2.2

.3.4

, 5

00

04

11

45

6

+

cros

s 4

.2,5

.8

reac

tion

s 6.

8, 5

3 kb

V

IB 1

88

LM

G 1

3215

D

./llb

rax,

Spa

in

cros

s 17

2

2 3.

3,

5 10

0412

456

+ reaction~

12

3 k

h V

IB 1

89

LM

G 1

3216

ro

tife

rs,

Gre

ece

5 17

2

2 88

kb

.3 3

24

45

37

57

+

V

IB 1

90

LM

G 1

3217

ro

tile

rs,

Gre

ece

5 17

2

2 88

kb

3 12

4413

757

+

VIB

19

1 L

MG

132

18

roti

fers

, G

reec

e 17

cr

oss

18

2 2

50

kb

3 3

24

41

27

57

+

re

acti

ons

VIB

19

2 L

MG

132

19

roti

fers

, G

reec

e 7

19

3 2

-19

0 k

b 6

30

46

10

55

6

+

VIB

193

L

MG

132

20

roti

fers

, G

reec

e 7

21

3 2

-20

0 k

b 6

32

46

10

55

6

+

VIB

194

L

MG

132

21

rori

fers

, G

reec

e 10

20

2

2 0

6 3

24

61

05

56

+

V

IB 1

95

LM

G 1

3222

A

rlem

ia,

Spa

in

7 V

aNT

1 21

2

2 0

6 3

24

61

05

56

+

V

IB 1

96

LM

G 1

3223

ro

tife

rs,

Gre

ece

0 2

2 °

1

32

47

72

55

7

+

VIB

197

L

MG

132

24

roti

fers

, G

reec

e V

aNT

7 I

2 16

kb

I

32

47

72

55

7

+

VIB

198

L

MG

132

25

roti

fers

, G

reec

e V

aNT

7 1

2 16

kb

32

47

72

55

7

+

VIB

199

L

MG

132

26

wat

er,

Gre

ece

VaN

T7

2

16

,49

, 3

24

77

25

57

11

+

68

kh

VIB

200

L

MG

132

27

Sp. a

ural

a. G

reec

e V

aW

7

2 16

kb

3

24

77

25

57

10

+

V

IB 2

01

LM

G 1

3228

Sp

. aum

ta,

Gre

ece

VaN

T7

2 16

kb

32

47

72

55

7

10

+

VIB

202

L

MG

132

29

D./

abra

x, G

reec

e V

aNT

7 2

16

,49

, 3

24

77

25

57

11

+

6

8,8

0 k

b V

lB 2

03

LM

G 1

3230

Sp

. aur

ata,

Spa

in

06

6

2 3

.2,5

.3,

32

47

52

75

7

16

+

50,

70 k

b V

IB 2

04

LM

G 1

3231

D

.labr

ax.

Spa

in

6 6

3 0

32

47

52

65

7

22

+

VIB

205

L

MG

132

32

wat

er,

Spa

in

06

(w

eak)

6

6 0

32

47

52

75

7

22

+

VIB

206

L

MG

132

33

roti

fers

, S

pain

0

6

6 6

2 0

32

47

52

65

7

22

+

VlB

207

L

MG

132

34

roti

lers

, G

reec

e 19

2

2 36

kb

6 3

24

61

05

56

+

V

IB 2

08

LM

G 1

3235

D

./abr

ax,

Gre

ece

20

2 2

36

kb

6 3

04

61

25

56

+

V

IB 2

09

LM

G 1

3236

D

. /a

brax

, G

reec

e 1

19

2 2

0 6

32

46

10

55

6

+

VIB

21

0

LM

G 1

3237

w

ater

, G

reec

e 7

wea

k 18

2

2 0

6 3

24

61

05

56

+

(w

eak)

cr

oss

reac

tion

s V

IB 2

11

LM

G 1

3238

w

ater

, G

reec

e 7

05

(w

eak)

20

2 2

120

kb

6 3

24

61

25

56

+

V

IB 2

12

LM

G 1

3239

D

./ab

rax,

Gre

ece

9 19

9

2 3

7 k

b 6

32

46

10

55

6

VIB

213

L

MG

132

40

wat

er,

Spa

in

13

19

17

2 4.

4 k

b

3 7

24

41

37

56

+"

V

IB 2

14

LM

G 1

3241

w

ater

, Gre

ece

18

04

26

15

2

49 k

b 5

15

61

25

57

+

"

Tab

le l

. C

onti

nued

Lab

orat

ory

Col

iect

ion'

l So

urce

and

/or

coun

try

Ribotyp~

Scro

-L

l'S

OM

I'

FAM

E

Plas

mid

B

IOL

OG

A

PI2

0E

Bio

type

I'a

tho-

refe

renc

e no

. st

rain

no.

of

ori

gin

clus

ter

grou

p pr

ofil

e pr

ofil

e pr

ofil

e co

nten

t pr

ofil

e pr

ofil

e pr

ofil

e ge

nici

ty

VIB

215

L

MG

132

42

D.la

br<

lx,

Spai

n 11

V

aNT

5 27

2

2 39

kb

6 30

4614

556

+ V

IB 2

16

LM

G 1

3243

D

.lab

rax,

Spa

in

VaN

T4

49

2 2

0 32

4752

757

15

+

VIB

217

L

MG

132

44

D.la

brax

, Sp

ain

VaN

Tl

49

2 2

0 1

3247

5275

7 +

V

IB 2

18

LM

G 1

3245

D

.lab

rax,

Spa

in

16

28

3 2

0 3

3006

5655

6 +

VIB

219

L

MG

132

46

roti

fers

, Sp

ain

06

2

6 2

0 1

3247

5275

7 +

V

IB 2

20

LM

G 1

3247

w

ater

, G

reec

e 1

1 2

0 1

3247

7255

7 11

+

VIB

221

L

MG

132

48

wat

er,

Gre

ece

1 2

2 0

1 32

4752

557

+ V

IB 2

22

LM

G 1

3249

D

.lab

rax.

Spa

in

06

6

1 2

44 k

b 32

4752

557

+

VIB

223

L

MG

132

50

Sp. a

llra/

a, G

reec

e 7

21

5 2

ISS

kb

6 32

4610

556

+

VIS

224

L

MG

132

51

D.l

abra

x, G

reec

e 8

05

21

11

2

0 6

3246

1255

6 +

VIB

225

L

MG

132

52

wat

er,

Gre

ece

2 21

7

2 14

7 kb

6

3246

1055

6 +

VIB

226

C

IP G

83.0

1 Sc

. max

imus

, Fr

ance

1

01

J

2 2

67 k

b 32

4752

657

+ V

IB 2

27

CII

'P03

.82

$(. m

axim

us,

Fran

ce

1 0

1

2 2

67 k

b 30

4752

677

+b

VIB

228

C

I1'1

9.22

Sp

. allr

a/a,

Fra

nce

01

1

67 k

b 32

4752

657

+ V

IB 2

29

clr 4

08F

O

. myk

iss.

Fra

nce

01

1

2 67

kb

3247

5275

7 +

V

IB 2

30

CI1

'1'0

9.80

O

. myk

iss,

Fra

nce

01

2

67

,78

kb

-32

4752

757

+ V

IB 2

31

err

07.

84

O. m

ykis

s, I

taly

01

I

67 k

b 32

4752

757

+ V

IS 2

32

ell' 0

4.88

O

. myk

iss,

Ira

ly

01

6

7k

b

3247

5265

7 +

VIB

233

ell

' 03.

88

O. m

ykis

s, I

taly

0

1

67 k

b 32

4752

757

+ V

IB 2

34

eiP

VA

408

trou

t, F

ranc

e 0

1

67 k

b 32

4752

757

+

VlB

235

el

l'V

62

D

.lab

rax,

Fra

nce

01

2

67 k

b 30

4752

457

+ V

IB 2

36

CIP

102

83

Sc. ,

nax

imll

s, F

ranc

e 1

01

2

2 67

kb

3247

7265

7 21

+

VIB

237

C

II'I

04

82

D

.lab

rax,

Fra

nce

I 0

1

2 3

6,6

7 k

b 30

4772

457

+b

VIB

238

el

P 1

0466

Sc

.max

;mIl

S, F

ranc

e 1

01

2

2 67

kb

3047

5265

7 21

+

0:

V

IB 2

39

CIP

106

21

sea

perc

h, G

reec

e 6

21

2 2

0 6

3246

1455

7 rc

+

~ V

IB 2

40

elP

/07

12

D.l

abra

x, M

orro

co

1 18

2

0 5

2246

5265

7 +

(w

eak)

n'

V

IB 2

41

ell

' 11

331

sea

perc

h, F

ranc

e 1

cros

s 2

1 2

0 1

3047

5247

7 +

~

reac

tion

s 0 ::l

V

IS 2

42

CIl

'1l1

64

Sc

. max

im liS

, Sp

ain

02

a 2

1 2

0 32

4752

757

20

+

co V

IB 2

43

NC

MB

187

5 O

. ner

ka,

U.S

.A.

01

&

2 5.

3, 1

1.5,

32

4772

757

26

+ ::

l 0

-

VaN

Tl

67 k

b -l

'<

V

IB 2

44

NC

MB

571

O

. myk

iss,

Jap

an

01

2

2 67

kb

3047

3275

7 +

'<

l

VIB

245

L

ee,

VL

3355

un

nam

ed f

ish,

U.K

. 0

1

2 2

67,

78 k

b 32

4772

657

+ S ""

VlB

246

H

WU

BA

136

Sa. t

rutta

, Ir

aly

01

2

2 67

kb

3247

5275

7 +

2.,

VlB

247

R

VA

U 1

989

salm

onid

fis

h, F

inla

nd

01

3

67 k

b 32

4772

557

30

+ <

V

IB 2

48

RV

AU

199

0 O

. myk

iss.

Fin

land

0

1

3 2

67 k

b 32

4752

757

23

+ 5- ...

VIB

249

R

VA

U 1

990

O. m

ykis

s, F

inla

nd

01

3

67 k

b 32

4752

757

+

<5

VIB

250

R

VA

U 1

990

O. m

ykis

s, F

inla

nd

01

3

67 k

b 32

4752

457

30

+ .. ~

VIB

251

C

arso

n 85

1352

6 O

. myk

iss,

Tas

man

ia

01

2

67 k

b 32

4752

757

15

+b

E.

VIB

252

C

arso

n 86

1383

1-2

O. m

yk;s

s, T

asm

ania

0

1

28

67 k

b 30

4752

757

+b

if

VIB

253

C

arso

n 85

/395

4-4

O. m

ykis

s, T

asm

ania

0

1&

2

67 k

b 32

4752

757

+b

... 1:

VaN

Tl

;:s

VIB

254

C

arso

n 89

/362

0 O

. myk

iss,

Tas

man

ia

01

2

67 k

b 9

3247

5275

7 18

+

VIB

255

C

arso

n 89

1287

3 O

. 11l

ykis

s, T

asm

ania

O

J 67

kb

2 30

4752

757

26

+ V

IB 2

56

Car

son

89/4

046

O. m

ykis

s. T

asm

ania

0

1

67

kb

1

3247

5275

7 15

+

N '"

VIB

257

C

arso

n 89

1066

O

. myk

iss,

Tas

man

ia

01

67

kb

I 32

4752

757

+

w

Tab

le 1

. C

on

tin

ued

t"

-c

.,. L

abor

ator

y C

olle

ctio

n'/

Sour

ce a

nd/o

r co

untr

y R

ibot

ype

Sero

-LP

S O

MP

FA~IE

Plas

mid

B

IOL

OG

A

PI2

0E

Bio

type

P

atho

-re

fere

nce

no.

stra

in n

o.

of

orig

in

dust

er

grou

p pr

ofil

e pr

ofil

e pr

ofil

e co

nten

t pr

ofil

e pr

ofil

e pr

ofil

e ge

nici

ty

?"

VIB

258

C

arso

n 89

/385

9 O

. myk

iss,

Tas

man

ia

01

1

67 k

b 1

3247

5275

7 15

+

;>

t:

V

IB 2

59

Car

son

89/4

068

O. m

ykis

s, T

asm

ania

O

J 1

67 k

b 2

3047

5275

7 15

+

'" ..

VIB

260

C

arso

n 89

/367

4 Sa

. saf

ar.

Tas

man

ia

01

1

2 67

kb

1 30

4752

757

+b

:; V

IB 2

61

Car

son

89/4

246

Sa. s

afar

, T

asm

ania

0

1

1 2

67 k

b 1

3247

5275

7 15

+

g

VIB

262

C

arso

n 89

/425

6 Sa

. saf

ar, T

asm

ania

0

1

1 67

kb

9 30

4752

757

'" 15

+

-

VIB

263

C

arso

n 89

/425

7 Sa

. saf

ar,

Tas

man

ia

01

1

67 k

b 30

4752

757

15

+

VIB

264

C

arso

n 89

/444

7 S,1

. saf

ar,

Tas

man

ia

01

1

67 k

b 32

4752

757

I, 1

5 +

V

IB 2

65

Car

son

89/3

997-

1 ro

tife

rs,

Tas

man

ia

0 0

3247

5277

7 15

+

V

IB 2

66

Car

son

89/3

748-

1 ro

tife

rs,

Tas

man

ia

VaN

TI

49

0 32

4652

757

15

+

VIB

269

E

vely

n, C

l O

. ket

a, C

anad

a V

aNT

I 1

5.3,

11.

5,

3044

7355

7 +

67

kb

VIB

270

E

vely

n, R

IP

O. t

shaw

ytsc

ha,

Can

ada

01

2

67 k

b 32

4772

757

+

VIB

271

L

MG

135

83

O. t

shaw

ytse

ha,

U.S

.A.

01

2

67 k

b 32

4752

657

+

VIB

272

L

MG

135

84

Japa

n 0

3

3 0

3247

5275

6 +

V

IB 2

73

LM

G 1

3585

O

. myk

iss,

U.S

.A.

01

2

2 67

kb

3244

5265

7 +

V

IB 2

74

LM

G 1

3586

O

. myk

iss,

Ita

ly

01

3

67 k

b 32

4726

57

+

VIB

275

L

MG

136

35

Cha

n os

chan

as,

Tai

wan

0

1

2 2

67 k

b 32

4772

657

+

VIB

276

L

MG

136

36

C. c

hano

s, T

aiw

an

01

2

67 k

b 32

4752

657

32

+

VIB

278

L

MG

139

48

U.S

.A.

Ot

2 67

kb

3247

7265

7 +

!

Typ

e st

rain

. 3

Sour

ces:

AT

CC

, A

mer

ican

Typ

e C

ultu

re C

olle

ctio

n, R

ockv

ille

, M

aryl

and;

AV

L,

Aqu

acul

ture

Vac

cine

s L

imit

ed,

Saf

fron

Wal

den,

Eng

land

; C

IP,

Col

lect

ion

of

the

Inst

itut

Pas

teur

, Pa

ri"

Fra

nce;

HW

U,

Her

iot-

Wat

t U

nive

rsit

y, E

dinb

urgh

, S

cotl

and;

LM

G,

Lab

orat

oriu

m v

oor

Mic

robi

olog

ie,

Rij

ksun

iver

site

it,

Gen

t, B

elgi

um;

NC

MB

, N

atio

nal

Col

lect

ion

of

Mar

ine

Bac

teri

a, A

berd

een,

Sco

tlan

d; R

VA

U,

Roy

al V

eter

inar

y an

d A

gric

ultu

ral

Uni

vers

ity,

Cop

enha

gen,

Den

mar

k; U

B,

Uni

vers

ity o

f B

arce

lona

. B

arce

lona

, S

pain

; D

r. j

. C

arso

t/, M

ount

P

leas

ant

Lab

orat

orie

s, K

ings

Mea

dow

s, T

asm

ania

, A

ustr

alia

; D

r. T

. P. T

. Eve

/yn,

Dep

artm

ent

of

Fish

erie

s an

d O

cean

s, P

acif

ic B

iolo

gica

l S

tati

on,

Nan

aim

o, B

ritis

h C

olum

bia,

Can

ada;

D

r. J.

V. L

ee,

Publ

ic H

ealt

h L

abor

atot

y, U

nive

rsity

Hos

pita

l, N

otti

ngha

m,

Eng

land

. b

Mor

ibun

d fis

h di

spla

yed

surf

ace

haem

orrh

ages

, pa

rric

ular

ly o

n th

e he

ad.

3. Serology

The majoriry (184/260 = 71 %) of the cultures, received as V. anglli//arum, were recovered in serogroups .01 -010, of which serogroup 01 with 95 isolates dommated (Table 1). Twenty isolates were considered to represent five previously undescribed serogroups, and are referred to

as VaNTl VaNT2, VaNT4, VaNT5 and VaNT7. A further con~plication concerned 16 cultures, of which 15 reacted with antisera representing two serogroups, and one was self-agglutinating (Table 1). The remaining 40 cultures did not react with any of the antisera used or cross reacted with more than two different antisera, and were regarded as untyped. It is noteworthy that the majority (24/40 = 60%) of the untyped isolates were not denved from fish but from anemia, rotifers, sediment and water.

Apart from serogroups 01 and 02a and 02b, which accommodated 134 (= 51 %) of the isolates from many fish species and geographical locations, smaller numbers of cultures were associated with serogroups 03 - 010 and VaNTl - VaNT7. Again, serogroups 04 and 05 with 9 and 11 isolates respectively, contained cultures obtained from a wide range of countries and habitats. In cont~ast, the 12 isolates recovered in serogroup 03 were obtamed only from fish in Denmark, Italy and Japan, and water from Denmark (Table 1). Isolates labelled as serogroups 06 and 08, with 6 and 4 isolates respectively, were ob­tained from many habitats in Europe. In particular, the isolates allocated to serogroup 06 were derived from Den­mark and Spain. The cultures regarded as serogroup 08 were obtained from Denmark and Italy. Five isolates, re­covered from fish (ayu [Plecoglossus altivelis], eel [Anguil­la anguilla], sea bream [Sparus aurata] and turbot [Scoph­thalmus maximus] in Denmark, Italy and Japan, belonged to serogroup 07. Three isolates from cod (Gadus morhua) obtained from Denmark were equated with serogroup 09 (2 isolates) and 010 (1 isolate) (Table 1).

All of the cultures, typed in serogroups 01, 02b, 03, 06,07,08,09,010, VaNT2 VaNT5 and VaNt:7, were recovered in cluster 1 as defined by ribotyping. It IS relev­ant to note that VIB 76, which was regarded as represent­ing serogroup 02a, was recovered as ribotype cluster 4; two isolates (VIB 185 and VIB 214) from serogroup 04 comprised riborype clusters 19 and 18, respectively; three cultures (VIB 23, VIB 211 and VIB 224) of serogroup 05 were defined as ribotype clusters 12, 7 and 8, respectively; and VIB 195 (serogroup VaNTI) and VIB 215 (serogroup VaNT5) were recovered in ribotype group 7 and 11, re­spectively. The remaining cultures ~ere unrypeable or de­monstrated serological cross reactions between several serogroups. Yet, these were mostly found within ribotype cluster 1.

4. Lipopolysaccharide (LPS) profiles

There was good agreement between the composition of serogroups 01, 02, 04, 06, 07, 08, 09 and 010 and LPS profiles. Thus, all of the isolates in serogroup 01 were homogeneous in terms of their LPS profile, being equated with LPS profile 1. Interestingly, 12 isolates in LPS group 1 were recovered in serogroup 03, 07, VaNT1 and

Identification and Typing of Vibrio anguillarum 295

VaNT7 and as untypable. Serogroup 02, which was sub­divided into 02a and 02b by serology, was regarded as a single LPS grouping, namely LPS profile. 2. In additio~, some of the isolates (3130 = 10%), whICh clustered 111

serogroup 02a, were equated with <;>ther LPS groups, namely LPS profile 14, 21 and 42. Five Isolates, which were ~quated with LPS profile 2, were recovered in sero­groups OS, 06, VaNT2 and as untypable. It should be emphasized that these apparently ab.berant strams were recovered in cluster 1, as defined by nbotypmg (Table 1). Cultures ryped as serogroup 04 were recovered principal­ly in LPS profile 4. Again, a minority (2/9 = 22%) were labelled as LPS profile 16 and 26. Of these, VIB 185 (sero­group 04) was recovered as a distinct single member clus­ter by ribotyping. Seven isolates, recovered in serogroup 03 (VIB 60-63, VIS 132, VIB 148 and VIB 272), were considered to represent LPS profiles 1, 42 and 45 (Table 1). Four cultures (VIB 22, VIB 23, VIS 211 and VIB 224), regarded as representatives of serogroup 05.' were ~quated with LPS profiles 20, 21 and 24. Indeed, It IS qu~stlonable whether these isolates are bona fide representatives of V. anguillarum.

Overall most cultures with LPS profile 1 and 2 corres­ponded t~ serogroups 01 and 02a and b, respectively. Nevertheless, LPS profile 1 was also recorded for cultures in serogroup 03, 07, VaNTI and VaNT7. Similarly, LPS profile 2 was noted among cultures in serogroups OS, O~ and VaNT2. Also, the distribution of the other LPS profi­les traversed serogroup boundaries. Thus, it would appear that typing systems based on LPS profiles do not coincide exactly with serogroups. However, some serogroups, e.? 01 and 02, demonstrated a higher level of homology 111

terms of the LPS profile than others.

5. OMP profiles There was negligible agreement between the composi­

tion of the serogroups and groupings resulting from ex­amination of the OMP profiles. It is notable that sero­group VaNT7, in which the component organisms ~ere equated exclusively with OMP group 1, was charactenzed by the presence of a 34 kDa protein. Yet, overall, the 260 cultures were recovered in 19 groups based on OMP pro­files. Cultures from serogroup 01 were equated with 4 groups based on the OMP profiles (Table 1). f:Iere, the dominant OMP profiles were 1, 2 and 3, which were characterized by the presence of dominant proteins of 33-35, 36-38 and 39-41 kDa, respectively. Again, the cultures recovered in serogroup 02 (a and b), 03, 04, OS, 06 07 and 08 were heterogeneous in terms of their OMP pro'fiIes. With serogroups 09 and 010

7 there wereinsuffi­

cient cultures to make meaningful detailed compansons. It should be emphasized that representatives of the dominant OMP groups appeared in a wide range of serogroups. For example, OMP profile 1 was found in cultures from sero­groups 01, 02a, 02b, 03, 05,06, 07, 08, VaNTl, VaNT2 and VaNT7 (Table 1). It was apparent that most of the cultures, recovered in ribotype clusters 2-20, re­presented unique OMP profiles. For example, OMP pro­file 15 was only recorded from VIB 214, which represent­ed ribotype cluster 10 (Table 1).

296 B. Austin et al.

6. FAME profiles

The major fatty acids were identified as C12: 0, C12: 0 30H, C14: 0, C16: 0 iso, C16: I w7c and C16 : 0 (Table 2). From the numerical analysis of the data, all isolates, except for four, were recovered in cluster 2 as delineated at Euclidean distance .6.9. The farry acid content of the isolates was very variable (Table 2). Within cluster 2, one group of isolates, which belonged to BIOLOG cluster 6, were distinct from the other cultures insofar as they exhi­bited a significantly higher C12: 01C12: 0 30H ratio. However, no correlation could be found between pheno­typic or serological diversity and the FAME profiles.

Cluster 1, which comprised two cultures, namely VIB 22 (serogroup 05) and VIB 24 (untyped by serology), were also phenotypically distinct and were recovered in BIOLOG cluster 8. Another two isolates, i.e. VIE 25 and VIB 204, comprised cluster 3, and were aberrant from other isolates insofar as they lacked C16: 0 iso, and pos­sessed a higher C16 : 0 content. Nevertheless, these may be regarded as authentic V. anguillarum cultures when ex­amined by other methods.

7. Plasmid profiles

Approximately half (137/260 = 53%) of the isolates carried plasmids. Isolates belonging to serogroups 03, 07,08,09 and 010 and VaNT4 were empty of plasm ids (Table 1). However, the majority (86/95 = 90%) of isolates in serogroup 01 contained the -67 kb virulence plasmid, equivalent to pJMl (Crosa et aI., 1980), either singly in most cases, or in association with one or two other plasm ids of 26-105 kb. The 67 kb plasmid was also found in VIE 243 and VIB 253, which demonstrated cross reactions between serogroups 01 and VaNTI, and VIB 269, which was labelled as VaNT1. Although most (32139 = 82%) of the isolates in serogroup 02 (a and b) were empty of plasm ids, 7 cultures contained one or two plas­mids of 5->200 kb (Table 1). Again, the minority (2/9 = 22 %) of isolates recovered as serogroup 04 possessed single plasm ids of either 32 or 49 kb. From serogroup OS, 5 isolates (45%) contained one (4 isolates) or two (1 iso-

Table 2. Major fatty acids of Vibrio anguilla rum

Fatty acid

C12:0 C12:0 30H C14:0 C16:0 iso C16:1OJ7c C16:0 l:F3 LF7

Ratios:

C12:0/14:0 CI2:0/12:0 30H CI6:0/16:1 w7c

3.1-6.4 1.7-3.4 4.1-7.9 2.4-3.7

30.4-39 18.4-25.4 2.2-4.5 9.6--21.5

0.79 1.88 0.63

Cluster:

2

1.4-7.1 0.1-4.8 3.4-10.1 0.1-10.5

34.1-44.4 17.5-28.6

1.3-5.3 9.3-22.0

0.72 1.62 0.62

3

7.4-8.2 4.5-6.0 8.4-11.8 0

41.2-43.2 41.5-43.2 5.16-6.4 5.6-7.3

0.77 1.42 0.46

late) plasmids of 2.6-120 kb. Two cultures (33%) from serogroup 06 contained one (44 kb) or four plasmids (3.2-70 kb). Isolates from the new serogroups were most-

9

8

7

6

5 4

:3

2

100 90 80 70 60 so 40

Fig. 2. Simplified dendrogram based on the examination of BIOLOG data by Pearson product correlation and UPGMA clus­tering.

ly devoid of plasmids. However, two (40%) of VaNTl isolates comprised two (53 and 56 kb) and three (5.3, 11.5 and 67 kb) plasm ids, respectively. Again, 2 (40%) of the isolates from VaNT2 comprised single (11.5 kb) or two (68 and 83 kb) plasmids. A single (50%) isolate from VaNT5 contained a 39 kb plasmid. All the cultures from VaNT7 contained a 16 kb plasmid. In addition, VIB 199 and VIB 202 both contained a 49 and a 68 kb plasmid. The majority of the serologically untyped cultures were empty of plasm ids. Expressed in terms of ribotyping, it was apparent that cultures from ribotypes 3, 4, 6, 8, 10, II, 12, 14, 16 and 20 were empty of plasm ids. However, a wide range of plasmid profiles was recorded from some of the isolates (Table 1).

8. BTOLOG fingerprints

Nine clusters were delineated at 78% r among the 260 isolates, using the Pearson correlation coefficient (Fig.2). All isolates tested in the BIOLOG GN-microplates were positive for oxidation of dextrin, glycogen, N-aceryl-D­glucosamine, D-fructose, D-glucose and maltose. Con­versely, all isolates were negative for adonitol, D-arabitol,

Table 3. Tests differemiating the clus-Test: ters as defined after numerical analysis

using the Pearson correlation coeffi-cient and UPGlvlA clustering of BIOLOG fingerprints Negative control

Cyclodextrin N-acetyl-D-galactosamine L-arabinose Gentobiose m-inositol a-D-Iactose D-mannose j)-methylglucoside D-sorbitol mono-methyl-succinate Acetic acid D-gluconic acid Succinic acid Bromosuccinic Alaninamide acid L-alanine L-alanylglycine L-aspartic acid Glycyl-L-aspartic acid Glycal-L-glutamic acid L-histidine L-proline L-threonine Uridine Thymidine Glycerol D,L-a-glycerolphosphate Glucose-I-phosphate Glucose-6-phosphate

Identification and Typing of Vibrio anguillarum 297

erythrirol, L-fucose, lactulose, D-melibiose, D-raffinose, turanose, xylitol, formic acid, galactonic acid lactone, D­galacruronic acid, D-glucosaminic acid, y-hydroxybutyric acid, p-hydroxy-phenylacetic acid, itaconic acid, a-keto­valerie acid, malonic acid, D-saccharic acid, sebacic acid, L-leucine, L-phenylalanine, L-pyroglutamic acid, D,L-car­nitine, y-amino-butyric acid, urocanic acid, phenyl-ethyl­amine, putrescine, 2-amino-ethanol and 2,3-butanediol. The differentiating characters (which were set at a dis­criminating level of 89%) between each cluster has been given in Table 3. The clusters did not possess any unique single features permitting differentiation from all other groups. Nevertheless, the number of characters needed to be used to distinguish between each of the delineated clus­ters has been presented in Fig. 3. From this diagram, it is apparent that dusters I, 2, 8 and 9 could not be disting­uished from each other. Clusters 2 and 9 each comprised two isolates deemed to be from serogroup 01. These clus­ters were similar to the majoriry of V. anguillarum isolates studied by other methods.

In common with the results of riboryping, the majority of the isolates, particularly those regarded as belonging in serogroup 01 (= 90%) and including the type strain, were

Cluster:

2 3 4 5 6 7 8 9

.B

54 + 50 20 + + + 20 33 60

65 + 83 20 33 + 33 50 + 33 + 80 + 47 + 20 + 25 17 20 + 20 + + 83 + + + + + + + + 60 + 80 + + + 50 80 33 60 + 85 50 17 + 80 + 76 50 17 67 60 33 + + + + 50 80 67 20 + + + + 17 67 87 + + + 85 50 17 20 + 80 + 73 + 60 + 65 + 20 + + + + + 83 + 40 + + + + + + + + + + + + 68 + 67 + + + 50 20 50 + 33 60 53 + 33 + + + 67 + + + + 30 50 + + 67 + 85 50 33 + + + + 63 67 + + 67 + + + 33 + + + + + 84 + 20 + 60 + + + 67 40 80 + + + + + 33 60 80 + + + . '+' and '-' correspond [Q >89% and <11% of positive responses. respectively. The

numbers correspond to the percentage of positive responses.

298 B. Austin et al.

Cluster: 1 2 3 4 5 6 7 8 9

1 0 2 0 0 3 2 9 0 4 4 8 1 0 5 4 9 5 6 0 6 2 8 8 9 2 0 7 2 7 7 7 9 5 0 8 0 3 6 9 4 1 1 0 9 Q Q B 12 1a 1~ 9 :2 Q

Fig. ]. Number of tests differentiating the 9 clusters defined by examination of the BIOLOG fingerprints.

recovered in a single large cluster 1, which accomodated 205 (= 79%) cultures. This group had an intragroup homogeneity of 80%. Yet, the membership of BIOLOG cluster 1 was not identical to that of the ribotype cluster 1. Thus, 21 of the isolates from ribotype cluster 1 were re­covered in BIOLOG clusters 2, 4, 5, 6, 7,8 and 9 (Table 1). Of the other isolates in serogroup 01, these were equ­ated with BIOLOG fingerprint clusters 2, 4 and 9, whereas VIB 18 and VIB 28 were unclustered. Interestingly, BIOLOG cluster 1 contained representatives of all sero­groups.

Serologically untyped isolates, which were mostly re­covered from water and live food, i.e. Artemia and rotif­ers, were grouped in cluster 3. These isolates corresponded to ribotype clusters 5, 13, 16, 17 and 20. The four isolates in BIOLOG cluster 2 were equated with serogroups 02 and 09. Cluster 4 contained 5 isolates of untyped V. an­guillarum and representatives of serogroups 01 and 02a from Japan, U.K. and U.S.A. One of these isolates corres­ponded to ribotype cluster 4.

The three isolates of cluster 5 were obtained from sea bass, and were untyped by serology. One of these isolates belonged to ribotype cluster 15, whereas the other re­presented ribotype cluster l.

Most isolates in cluster 6 were isolated from Greece. Yet only a few of the isolates could be typed by serology, and were equated with serogroups OS, VaNTI and VaNT5. These isolates were dispersed among ribotype clusters 2, 6, 7, 8, 9, 10 and 11. Interestingly, the isolates in cluster 7 were recovered from water and rainbow trout (Oncorhyn­chlls mykiss; VIB 142) in Denmark. Although most of these isolates were untyped by serology, VIB 142 was re­garded as a member of serogroup VaNT2. Two of the isolates of cluster 7 constituted ribotype cluster 14.

A geographical pattern was also apparent with the ori­gin of the four isolates in cluster 8, being obtained from sea bass (Dicmtrarchus labrax) in Greece and Artemia in Spain. These isolates were either untyped or represented serogroups 04 and 05. Two of these isolates constituted ribotype clusters 12 and 19.

Overall, the isolates regarded as belonging to serogroup 02 were biochemically diverse, being distributed among BIOLOG clusters 1 and 4. Similarly, the isolates equated with serogroups 04, OS, 09, VaNT2 and VaNT5 were

recovered in many clusters based on BIOLOG profiles. Isolates, which were untyped by serology, were found in clusters 1,3,5,6,7 and 8.

9. API 20E profiles

Overall, the cultures of V. anguillarum were fermenta­tive, motile, produced arginine dihydrolase, I~-galactosid­ase, indole and oxidase, but not lysine or ornithine decarb­oxylase, degraded gelatin but no·t urea, reduced nitrate to nitrite but not to nitrogen, gave a positive response to the Voges Proskauer reaction, and produced acid from arabin­ose, mannitol and saccharose, but not from amygdalin, inositol or melibiose. Yet marked heterogeneity was re­corded in the individual API 20E profiles for V. anguil­farum. Thus, 117 different profiles were recorded, of which '324772757', '324752757', '324772557', '304752757' and '324772757' accounted for 112 (= 43%) of the isolates (Table 1). Generally, there was little relationship between these profiles and the composition of the serogroups. For example, the cultures of serogroup 01 were represented by 25 API 20E profiles, of which '324752757' was dominant. Yet, this profile was the most common, being recorded for 55 isolates representing 11 serogroups. Indeed, this API 20E profile predominated among the isolates of serogroups 03, 04 and 010 (Table 1). Thus, the API 20£ profiles permitted certain differenti­ation among the isolates of V. anguillarum. Yet, this dif­ferentiation could not be equated with the other typing methods used in this study. Nevertheless, it was apparent that the most extreme API 20£ profiles belonged to cul­tures which were equated with ribotypes other than 1. For example, the profile '000411456' was recorded from isolates of ribotype 15 (Table 1).

10. Biotyping

The minority (106/260 = 41 %) of the cultures were recovered in 32 clusters, which were defined at or above the 80% similarity level using the data for Biotype-100 (Table 1). The Biotype clusters were defined as groups of strains (or isolated strains) with identical (dissimilarity = 0) nutritional patterns. The remaining 154 isolates com­prised single members clusters, each with a separate Bioty­pe profile. However, these isolates did not use many sub­strates, possibly reflecting the lack of sufficient growth factors in the media. The differentiating characteristics be­tween the clusters have been included in Table 4. It was apparent that the majority of isolates utilized a wide range of carbon compounds in four days, including N-acetyl-D­glucosamine, L-aspartate (1 exception), D-fructose, D­gluconate (1 exception), D-glucose, L-glutamate, L-malate (1 exception), D-mannitol, D-mannose, 1-0-methyl-/3-D­glucoside, L-proline and sucrose. Conyersely, the isolates were uniformly negative in their ability to utilize trans­aconitate, adonitol, 4-aminobutyrate, 5-aminovalerate, D­arabitol, L-arabitol, benzoate, betaine, caprate, caprylate, m-coumarate, dulcitol, i-erythritol, aesculin, ethanol­amine, L-fucose, D-galacturonate, gentobiose, gentisate, D-glucuronate, glutarate, histamine, L-histidine, 3-hy­droxybenzoate, 4-hydroxybenzoate, 3-hydroxybutyrate,

Idenriiication and Typing of Vibrio angllillamm 299

Table 4. Tests diiferenriating the c1usrers as defined by use of biotyping

Carbon conraining compound·':

Cluster:

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

4

+ + + + + + + + + + + + + + + +

+

+ + + + + + + +

14

+

+

+

+ +

16

+ + + +

+ + + + + + + + + + + + + + + + + +

20

+

+

+ + + + +

+ + + + + + +

+ + + + + + + + + + + + +

21

+ + +

+

+ +

+

+ +

+ + + + + + + + +

+

+

+ + + +

37

+ + + +

+ + + + + + + + + + + + + + + + + + + + +

+ +

49

+ +

+ +

+ +

76

+ + + + + + + + + + + + + + + + + +

+ + + +

+ + + + + +

93

+ + + + + + +

+ + + +

+ + + +

+ + +

+

+ + +

+

94

+ + + + + + + + + + + + +

+ + + +

+ + +

+

+ + +

95

+ + + + + + + + + + + + +

+ + + +

+ + + + + + + + + + + +

98

+ +

99

+ +

+ +

+

a 4, 14, 16, 20, 21, 33, 37,49, 76, 93, 94, 95, 98 and 99 correspond to D-trehalose, 1-0-methyl-l3-galactopyranoside, D(+) cellobiose, D( -) ribose, L( +) arabinose, myo-inositol, D-sorbitol, D( +) malate, DL-Iactate, D-alanine, L-alanine, L-serine, L-serine, L­tyrosine and a-ketoglutarate, respectively.

hydroxyquinoline-B-glucuronide, itaconate, 2-ketogluco­nate, 5-ketogluconate, lactose, lactulose, D-Iyxose, malo­nate, maltitol, D-melezitose, D-melibiose, 1-0-methyl-a­galactoside, 3-0-methyl-D-glucose, 1-0-methyl-a-D­glucoside, mucate, palatinose, phenylacetate, 3-phenyl­propionate, propionate, protocatechuate, putrescine, quinate, D-raffinose, L-rhamnose, D-saccharate, L-sor­bose, D-tagatose, D-tartrate, L-tartrate, meso-tartrate, tricarballylate, trigonelline, tryptamine, tryptophan, D­turanose, xylitol and D-xylose. Differential results, In

terms of the % positive response to the utilization of the carbon containing compounds, was recorded for cis­aconitate (91 %), D-alanine (72%), L-alanine (75%), L­arabinose (60%), D-cellobiose (77%), citrate (94%), fumarate (91%), D-galactose (98%), D-glucosamine (89%), DL-glycerate (91 %), glycerol (97%), myo-inositol (9%), 2-ketoglutarate (8%), DL-Iactate (83%), D-malate (14%), maltose (95%), maltotriose (96%), 1-0-methyl-f3-

galactoside (6%), D-ribose (86%), L-serine (85%), D-sor­bitol (91 %), succinate (95%), D-trehalose (72%) and L­ryrosine (2%).

11. Pathogenicity

The majority (226/260 = 87%) of the isolates were pathogenic to salmonids at a dose of 105 - 106 cells/fish, as determined by the development of overt clinical signs of disease and evenrual mortalities. Overall, high proportions of the isolates from serogroup 01 (96%), 02a (93%), 02b (100%), 03 (75%), 06 (100%), 07 (80%), 08 (100%), 10 (100%), VaNTl (100%), VaNT4 (100%), YaNT5 (100%) and VaNT7 (100%) and unrypable (83 %) were pathogenic. In contrast, less isolates from serogroup 04 (55%), 05 (54%),09 (40%) and YaNT2 (40%) demonstrated pathogeniciry. Of the non-patho­genic culrures, only 4 (12%) contained plasmids. Thus,

300 B. Austin et al.

VIB 88, VIB 93, VIB 166 and VIB 212 contained 67 kb and 105 kb, 67 kb, 2.6 kb, and 37 kb plasmids, respective­ly. It should be emphasised that other cultures, which did not contain plasm ids, were pathogenic. By comparing pathogenicity with the ribotype, it was apparent that 100% of the isolates from ribotype 2-8 and 10-20 were pathogenic. In contrast, pathogenicity levels of 98% and 0% was recorded for the isolates from ribotype 1 and 9, respectively. Generally, the pathology was consistent with septicaemia, namely the development of gastro-enteritis, ascites, haemorrhaging in the musculature, and liquefac­tion of the internal organs, especially the kidney. Some cultures (total = 26), representing a diverse range of sero­groups and origin, resulted in the development of exten­sive surface haemorrhaging, particularly on the head and in the mouth (Table 1).

Discussion

Overall, this study sought to clarify the identificationl typing of isolates, considered to be bona fide represen­t,l[ives of V. anguillarum. The strengths of the investiga­tion centred on the large collection of bacterial strains, which were obtained from a diverse range of geographical locations, hosts and environmental samples. However, the influence of the different methods of analyses of the data obtained in this study is largely unknown (Priest and Au­stin, 1993). In contrast to previous phenetic based studies, e.g. Kaper et al. (1983), the bulk of these isolates were recovered in a single homogeneous group, as defined by ribotyping. Undoubtedly, this group (cluster 1) constituted authentic isolates of V. angui/larum. It is accepted that the other clusters as defined by ribotyping, i.e. clusters 2-20, may not, in fact, be V. anguillarum. The taxonomic posi­tion of these small number of isolates must await further investigation. Yet, it was apparent that the various methods did not give congruent results. For example, some of the other clusters defined by ribotyping could be equated with existing serogroups of V. anguillamm. In short, the various methods enabled recognition of core members of the taxon, but there was a problem with boundary definition. The greatest discrepancies occurred with the non-typed isolates (by serology), some of which must be considered to be on the periphery of the species. In contrast, the highest congruence in results occurred with the bulk of the isolates, which were considered to repre­sent serogroup 01. This is arguably the best understood of all the subgroups of V. allguilfarum (Pedersen and Larsen, 1993; Austin and Austin, 1993). Thus, the majority of these isolates belonged to LPS group 1, possessed the 67 kb plasmid, which was found to be associated with viru­lence (Crosa et aI., 1980), were recovered in the same BIOLOG cluster, i.e. 1, and were highly pathogenic to fish. However, a small number of isolates from serogroup 01 possessed the virulence plasmid but were not pathogenic. Moreover, some isolates were pathogenic and did not possess the 67 kb plasmid. Thus, in contrast to previous studies, it is apparent that there is not an absolute correlation between serogrouping and LPS profiles (Aoki

et aI., 1981) and plasmid profiles and virulence (Crosa et aI., 1980).

The validity of separating serogroup 02 into 02a and 02b must be questioned in view of some of the data from this study, notably the results of the LPS analysis which could not distinguish between the sub-groups. This has also been reported by Mutharia et al. (1993).

The LPS of Gram-negative bacteria corresponds to the O-antigen, and inevitably forms the basis of serogrouping. Therefore, a close relationship between serogroup and LPS profile may usually be anticipated. However in the present study, this association was most evident with certain sero­groups, i.e. 01, 02, 04, and 06-010. With the other serogroups, the correlation to LPS profile was lower. In­deed, in serogroups 03, VaNTl, VaNT4 and VaNT5, the isolates belonged to separate LPS profiles. An interpreta­tion of these results is that certain LPS antigens have im­munological cross-reactivity. Alternatively, the bacteria may contain common heat-stable antigens, other than LPS. This possibility needs further investigation. Clearly, serogrouping is not always adequate for predicting the nature and composition of the LPS of V. anguilla rum, insofar as some isolates revealed identical typing patterns with the O-antisera but possessed different LPS profiles. Overall, the pool of serologically non-typable isolates were the most diverse in terms of LPS profiles. This indicates that there are additional serogroups still to be defined. Interestingly, only 78% of the isolates, examined in this study, could be typed by serology, which is a lower pro­portion than previously reported (Larsen et aI., 1994). A possible explanation may reflect the comparatively large number of environmental isolates examined here in com­pari~on to previolls studie~.

The discriminatory power of plasmid profiling was gen­erally low. Therefore, it is conceded that plasmid profiles are of minor value for typing V. anguillarum. An increased discriminatory value of plasmid patterns, particularly for representatives of serogroup 01, may be achieved by re­striction enzyme analysis of isolated plasmids (Olsell and Larsen, 1990). Certainly, the association of the 67 kb plas­mid with serogroup 01 is in agreement with previous work (Wolf and Crosa, 1986). The presence of this plasmid in the previously undescribed serogroup, coined VaNTl, and the frequent cross-reaction between serogroups 01 and VaNTl, raises the possibility that the new group is merely a variant/sub-group of serogroup 01. This needs further investigation. The presence of a 67 kb plasmid encoding an iron sequestering mechanism, consisting of a siderophore and an outer membrane protein, OM2, has long been considered to explain the virulence of isolates of serogroup 01 (Crosa, 1980), with curing of the plasmid leading to a marked increase in LDso values (Crosa et aI., 1980). Yet, it should not be overlooked that in the present study nine isolates, equated with serogroup 01, were either non- or weakly pathogenic. Three isolates contained the 67 kb plasmid. The reason for the lack of virulence by these few cultures is unclear. Conceivably, the iron seques­tering mechanism may not have been expressed.

The pathogenicity of V. anguilla rum for fish, especially salmonids, is well established (Austin and Austill, 1993).

The association of some serogroups, i.e. 01, 02 and 03, with epizootics has been reported (S(Jrensen and Larse'l, 1986; Larsen et aI., 1994). Indeed, the data from this study have highlighted the virulence of representatives of these serogroups. An inference that other serogroupS are mostly non-pathogenic and comprise environmental isolates is partially supported by this study, insofar as only approximately half of the cultures from serogroups 04, OS, 09 and YaNT2 caused mortalities in Atlantic salmon. Of course, it may be argued that the apparently non­pathogenic isolates could well cause disease in other fish species under less favourable environmental conditions (= stress) (Austin and Austin. 1993).

Overall, the number of profiles obtained with the API 20E rapid identification system greatly exceeded previous studies (Kent, 1982; Maugeri et aI., 1983; Grisez et aI., 1991), and, together with the results of biotyping demons­trated the biochemical heterogeneity of V. anguillarum. Yet, this was not so clear with BIOLOG, which revealed a smaller number of profiles. It should be emphasised that caution needs to be exercised when using systems designed for other uses. Thus, the API 20E system was originally developed for medically important isolates of the family Enterobacteriaceae, which grew at 37°C within 48 h. Yet for non-human isolates, notably V. anguillarum as used in this study, lower incubation temperatures often need to be adopted. Thus, the comparison of data to schemes de­signed for use with medically important bacteria may well give rise to erroneous identifications. However, a good identification of V. anguillarum has been achieved by us­ing the results of the API 20E rapid identification system with specially formulated keys for vibrios (Alsina and Blanch, 1994a, b).

It will remain for further work to clarify the precise taxonomic position of V. anguilla rum and its relationship to the genus Listonella. Yet, the validity of the genus Lis­tonella has been thrown into doubt when V. damsela was reclassified to Listonella (MacDonell and Colwell, 1985) and thence to Photo bacterium (Smith et aI., 1991). V. anguillarum has been considered to be more closely related to V. pelagius than to organisms with the specific epithet of damsela in terms of ribotyping (Olsen and Larsen, 1993). The current view from this study is that strains with the specific epithet of anguillarum would be better suited in the genus Vibrio, as V. anguillarum.

As a result of this study, the characteristics of typical isolates of V. anguillarum may be amended from the de­scription in Baumann et al. (1984), to:

Gram-negative, motile fermentative, rod-shaped cells, which produced arginine dihydrolase, catalase, f)-galac­tosidase, HlS, indole and oxidase but not lysine or or­nithine decarboxylase or tryptophan deaminase, reduced nitrate to nitrite but not to nitrogen, were positive for the Yoges-Proskauer reaction, degraded gelatin but not urea, utilized citrate, and produced acid from amygdalin, arabinose, glucose, sorbitol and sucrose but not from in­ositol, mannose, melibiose or rhamnose. Dextrin, D-fruc­tose, glycogen, N-acetyl-D-glucosamine, D-fructose, mal­tose and sucrose were utilized, but not so adonitol, D­arabitol, erythritol. L-fucose, lactulose, D-mannose, D-

Identification and Typing of Vibrio anguillarum 301

melibiose, D-raffinose, turanose, xylitol, formic acid, galactonic acid lactone, D-galacturonic acid, D­glucosamininic acid, y·hydroxybutyric acid, p-hydroxy­phenylacetic acid, itaconic acid, a-keto-valeric acid, malonic acid, D-saccharic acid, sebacic acid, L-leucine, L­phenylalanine, L-pyroglutamic acid, D,L-carnitine, y­amino-butyric acid, urocanic acid, phenylethylamine, put­rescine, 2-amino-ethanol or 2,3-butanediol. Growth oc­curred on MacConkey agar. Cultures comprised a single dominant ribotype, but may be typed in any of a diverse range of serogroups, of which representatives of serogroup 01 may contain a 67 kb plasmid associated with viru· lence. Two dominant groups have been defined with LPS profiles. OMP were typically of 33-40 KDa in size. Pathogenicity was exhibited to fish.

Acknowledgement. Financial support was provided by CEC Contract No: AIR-Cf92-0341. We are grateful to colleagues for the gift of cultures.

References

Alsina, M., Blanch, A. R.: A set of keys for biochemical identifi· cation of environmental Vibrio species. J. Appl. Bacteriol. 76, 79-85 (1994a)

Alsina, M., Bla"ch, A. R.: Improvement and update of a set of keys for biochemical identification of Vibrio species. J. Appl. Bacterial. 77, 719-721 (1994b)

Aoki, T., Kitao, T., [tabashi, T., Wada, Y., Sakai, M.: Proteins and lipopolysaccharides in the membrane of Vibrio anguil­larum. Devel. BioI. Stand. 49, 225-232 (1981)

Austin, B., Austin D. A.: Methods for the Microbiological Ex­amination of Fish and Shellfish. Chichester, England, Ellis Horwood (1989)

Austin, B., Austin, D. A.: Bacterial Fish Pathogens Disease of Farmed and Wild Fish, 2nd edn. Chichester, England, Ellis Horwood (1993)

Baumann, P., Furniss, A. L., Lee, J. V.: Genus 1. Vibrio Pacini 1854, 411AL, pp. 518-538. In: Bergey's Manual of Systematic Bacteriology, Vol. 1 (N. R. Krieg, J. G. Holt, eds.). Baltimore, Williams & Wilkins (1984)

Crosa, J. H.: A plasmid associated with virulence in the marine fish pathogen, Vibrio anguillarum specifies an iron sequester· ing system. Nature (London) 284, 566-568 (1980)

Crosa,J. H., Hodges, L. L., Schiewe, M. H.: Curing of a plasmid is correlated with attenuation of virulence in the marine fish pathogen Vibrio anguillarum. Infect. Immun. 27, 897-902 (1980)

DeBoer, S. H., Sasser, M.: Differentiation of Erwinia carotovora ssp. carotovora and E. carotovora ssp. atroseptica on the basis of cellular fatty acid composition. Can. J. Microbiol. 32, 796-800 (1986)

Grimont, F., Chevrier, D., Grimont, P. A. D., Lefevre, M., Glles­don, J. L.: Acetyiaminofluorene-iabelled ribosomal RNA for use in molecular epidemiology and taxonomy. Res. Microbiol. 140,447-454 (1989)

Grisez, L., Ceusters, R., Ollevier, F.: The use of AP[ 20E for the identification of Vibrio anguilla rum and V. ordalii. J. Fish Dis. 14,359-365 (1991)

Hitchcock, P. J., Brown, T. M.: Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver­stained polyacrylamide gel. J. Bacteriol. 154, 269-277 (1983)

302 B. Austin et al.

Kado, C. T., Lill, S.-T.: Rapid procedure for detection and isola­tion of large and small plasmids. J. Bacterial. 145, 1365-1373 (1981)

Kaper, J. B., Lockman, H., Remmers, E. F., Kristensen, K., Col­well, R. R.: Numerical taxonomy of vibrios isolated from es­tuarine environment. Int. J. System. Bacterial. 33, 229-255 (1983)

Kent, M. L.: Characteristics and identification of Pasteurella and Vibrio species pathogenic to fishes using API-20E (Analytab Products) multitube test strips. Can. J. Fish. A'Iuat. Sci. 39, 1725-1729 (1982)

Kitao, T., Aoki, T., Fllkodome, M., Kawano, K., Wada, Y., MiZllno, Y.: Serotyping of Vibrio anguillarum isolated from diseased freshwater fish in Japan. J. Fish Dis. 6, 175-181 ( 1983)

Kittclberger. R .• Hilbink. F.: Sensitive silver-staining detection of bacterial lipopolysaccharides in polyacrylamide gels. J. Bio­chern. Biophys. Ivlethods 26,81-86 (1993)

Kllsuda, R .. Sako, H., Kawai. K.: Classification of vibrios iso­lated from diseased fishes - 1. On the morphological, biologi­cal and biochemical properties. Fish Pathol. /3, 123-137 (1979)

Lambert. P. A.: Isolation and purification of outer membrane proteins from Gram-negative bacteria, pp. 110-125. In; Bacte­rial Cell Surface Techniques (I. Hancock. I. Poxton. eds.). Chichester, England, John Wiley & Sons (1986)

Larsen, J. L., Olsen. J. E.: Occurrence of plasm ids in Danish isolates of Vibrio angllillarum serovars Oland 02 and associ­ation of plasm ids with phenotypic characteristics. Appl. Envi­ron. Microbiol. S7, 2158-2163 (1991)

Larsen. J. L.. Pedersen, K .• D'l/sgaard, 1.: Vibrio anguilla rum serovars associated with vibriosis in fish. J. Fish Dis. 17, 259-267 (1994)

Maniatis, T., Fritsch. E. F., Sambrook, J.: Molecular cloning; a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982)

MacDonell, M. T., Colwell, R. R.: Phylogeny of the Vibrionaceae and recommendations for two new genera, Listonella and Shewanella. System. Appl. Microbial. 6, 171-182 (1985)

MacDonell, M. T., Swartz, D. G., Ortiz-Conde, B. A., Last, G. A., Colwell, R. R.: Ribosomal RNA phylogenies for the vibrio­enteric group of eubacteria. Microbiol. Sci. 3, 172-179 (1986)

Maugeri, T. L., Crisafi, E., Genovese, L., Scoglio, M. E. R.: Identification of Vibrio angllillarum with the API-20E system. Microbiologica I, 73-79 (1983)

Merrill. C. R., Goldman, D., Sedman. S. A .• Ebert, M. H.: UI­trasensitive stain for proteins in polyacrylamide gcls shows regions in cerebrospinal fluid proteins. Science 21,1437-1438 (1981)

Muroga. K., Yama11Oi, H., Hiranaka, Y., Yamamoto, S .. Tatani. M .. Io, Y .• Takahashi, S., Hanada. H.: Detection of Vibrio

anguilla rum from wild fingerlings of ayu Plecoglosslis altive/is. Bull. Jap. Soc. Sci. Fish. 50, 591-596 (1984)

Mutharia, L. W., Raymond, B. T.. Dekie~'it. T. R .• Stevenson, R. M. W.: Antibody specificities of polyclonal rabbit and rainbow trout antisera against Vibrio ordalii and serotype 0; 2 strains of Vibrio angllillarum. Can. J. ~1icrobiol. 39, 492-499 (1993)

Nybelin, 0.: Untersuchungen iiber den bei Fischen krankheitser­regenden Spaltpilz Vibrio anguillarttm. Med. Stat. Undersok. Fors. Anst. Sotvat. Stockholm 8, 1-62 (1935)

Olsen, j. E., Larsen, j. L.: Restriction fragment length poly­morphism of the Vibrio anguilla rum serovar 01 virulence plasmid. Appl. Environ. Microbiol. S6, 3130-31>2 (1990)

Olsen, j. E., Larse11, j. L.: Ribotypes and plasmid contents of Vibrio anguillaru»l strains in relation to serovar. App!. Envi­ron. Microbiol. 59, 3863-3870 (1993)

Pedersen, K., Larsen, J. L.: rRNA gene restriction patterns of Vibrio anguilla rum serogroup 01. Dis. Aquat. Org. 16, 121-126 (1993)

Priest, F. G., Austin, B.: Modern Bacterial Taxonomy, 2nd. edn. London, Chapman & Hall (1993)

Rochelle, P. A., Fry, J. c., Day, M. j., Bale, M. J.: An accurate method for estimating sizes of small and large plasmids and DNA fragments by gel electrophoresis. J. Gen. Microbiol. 132, 53-59 (1985)

Smith, I. W.: A disease of finnock due to Vibrio anguillarum. J. Gen. Microbiol. 74, 247-252 (1961)

Smith, S. K., Sutton, D. C, Fuerst, J. A., Reichelt. j. L.: Evalua­tion of the genus Listonella and reassignment of Listonella damsela (Love et al.) MacDonell and Colwell to the genus Photobacterium as Photobacterium damsela comb. nov. with an emended description. Int. J. Syst. Bacteriol. 41, 529-534 (1991)

Sneath, P. H. A .• Johnson, R.: The influence on numerical tax­onomic similarities of errors in microbiological tests. J. Gen. Microbiol. 72, 377-392 (1972)

Sneath, P. H. A., Sokal, R. R.: Numerical Taxonomy: The Princi­ples and Practice of Numerical Classification. San Francisco, W. H. Freeman (1973)

Sorensen. U. B .• Larsen. j. L.: Serotyping of Vibrio anguillarum. Appl. Environ. Microbiol. SI, 593-597 (1986)

West, P. A., Lee, j. V., Bryant, T. N.: A numerical taxonomic study of species of Vibrio isolated from the aquatic environ­ment and birds in Kent, England. J. Appl. Bacteriol. SS, 263-282 (1983)

Wiik, R., Hoff. K., Andersen. K., Da,/e. F. L.: Relationship be­tween plasm ids and phenotypes of presumptive strains of Vib­rio anguilla rum isolated from different fish species. Appl. Envi­ron. Microbiol. 55, 826-831 (1989)

Wolf, M. K., Crosa, j. H.: Evidence for the role of a siderophore in promoting Vibrio anguillamm infections. J. Gen. Microbiol. 132,2949-2952 (1986)

Professor B. Austin (Department of Biological Sciences, Heriot-Watt University, Riccarron, Edinburgh EH14 4AS, Scotland UK)