1
1
2
Di e si y o Vib ios associa ed wi h ea ed clams in Galicia (NW3
Spain).4
5
Roxana Beaz Hidalgo1, Ilse Cleenwe ck2, Sabela Balboa1, Ma jan De Wach e 2,6
Fabiano L. Thompson3, Jean Swings2, Paul De Vos2 & Jesús L. Romalde1*7
8
1 Depa amen o de Mic obiología y Pa asi ología. Facul ad de Biología e Ins i u o de9
Acuicul u a. Uni e sidad de San iago de Compos ela. 15782. San iago de Compos ela.10
2 BCCM/LMG Bac e ia Collec ion, Labo a o y o Mic obiology, Ghen Uni e si y,11
Ghen , Belgium.12
3Depa men o Gene ics, Ins i u e o Biology, Fede al Uni e si y o Rio de Janei o13
(UFRJ), Rio de Janei o, B azil.14
15
16
17
18
19
20
21
22
Accep ed in: Sys ema ic and Applied Mic obiology, Ma ch, 200823
24
25
26
27
* Co esponding au ho :28
Phone: +34 981563100 # 1325329
Fax: +34 98159690430
E-mail: [email p o ec ed]31
32
33
2
ABSTRACT34
The aim o he p esen s udy was o cha ac e ize and iden i y ib ios isola ed om35
cul u ed clams in Galicia (NW Spain). A o al o 759 isola es we e ob ained,36
pheno ypically cha ac e ized, g ouped and assigned o he genus Vib io. Subsequen ly,37
he genomic di e si y o 145 ep esen a i e s ains was analyzed by means o ampli ied38
agmen leng h polymo phism (AFLP) and e ealed a high gene ic di e si y amongs 39
hose isola es. Only 57 ou o 145 s ains could be iden i ied o he species le el, which40
we e dis ibu ed in 13 AFLP clus e s. V. cycli ophicus, V. splendidus and V.41
alginoly icus we e he mos abundan ly ep esen ed species. Eigh y eigh isola es42
emained uniden i ied, 59 we e dis ibu ed o e 16 clus e s, while 29 we e unclus e ed.43
Sequencing o he 16S RNA and wo house-keeping ( poA and ecA) genes o 44
ep esen a i e s ains belonging o he 8 uniden i ied clus e s wi h highe numbe o 45
isola es con i med hei assigna ion o he Vib ionaceae amily, some o hem p obably46
ep esen ing new species wi hin he genus. The p esen s udy con i ms ha he47
pheno ypic cha ac e iza ion o ib ios is no enough o iden i y hem a species le el. A48
wide di e si y o ib ios was ound in cul u ed clams o all ou geog aphic loca ions49
analyzed. In o al, mo e han 12 Vib io species and a leas h ee po en ial new species50
in his genus we e iden i ied.51
52
Keywo ds: Vib ios, di e si y, clam, AFLP, sequencing.53
3
INTRODUCTION54
The cul u e o clams ep esen s an impo an ma ine esou ce and is o g ea economic55
impo ance o he coas o Galicia (NW Spain). Na u al clam beds o au hoc onous56
ca pe -shell clam (Rudi apes decussa us) ha e been ha es ed beyond hei maximum57
sus ainable yield, which has led o he in oduc ion o he o eing species Manila clam58
(Rudi apes philippina um) o co e he consume demands, wi h he isk o in oducing59
new pa hogenic s ains [20]. Pe iodically, mo ali ies occu in hese clam popula ions60
which, un il now, could no be de ine i ely associa ed o any in ec ious agen o 61
en i onmen al ac o .62
The amily Vib ionaceae is au och honous o aqua ic en i onmen s including es ua ine,63
coas al wa e s and sedimen s wo ld wide, and some species a e well known pa hogens64
o ma ine o ganisms including ish, co als and molluscs [8, 21, 24, 32]. Vib iosis65
mainly a ec s nu se y cul u es o ju enile bi al es. Vib ios associa ed wi h in ec ion in66
bi al e molluscs belong o he species such as Vib io ape is, Vib io c assos eae,67
Vib io alginoly icus and Vib io pec enicida [15, 17, 19]. In Spain, mass mo ali ies ha e68
been epo ed in adul clams due o V. ape is in ec ions [4], and in seed and ju eniles69
due o Vib io splendidus II, V. nep unius and V. alginoly icus [8, 20]. O he molluscs a e70
also suscep ible o in ec ion by ib ios including scallops Pec en maximus [17], oys e s71
C assos ea gigas [34] and abalone, Halio is di e sicolo supe ex a [16]. Howe e ,72
sca ce in o ma ion exis s on he occu ence o ib ios o any o he bac e ia ha a e73
pa hogenic o cul u ed clams [3,19].74
The main aim o his s udy is o analyze he Vib io popula ions associa ed wi h cul u ed75
clams in Galicia, using, a polyphasic app oach which include pheno ypic76
cha ac e iza ion, ampli ied agmen leng h polymo phism (AFLP) analysis and77
sequencing o he 16S RNA and wo house-keeping genes, in o de o de e mine he78
4
no mal mic obio a and also o be able o iden i y po en ial new pa hogens o hese79
bi al e molluscs.80
81
MATERIALS AND METHODS82
Sampling, bac e ial s ains and g ow h condi ions83
Cul u ed clams we e mon hly sampled om 4 geog aphic loca ions (A o D) in he84
A lan ic No hwes o Spain (Fig. 1) om Ma ch 2004 o Sep embe 2005. O ganisms85
we e anspo ed ali e on ice o he labo a o y and analyzed wi hin 3 hou s. A o al o 86
15-20 clams, depending on he size, we e asep ically dissec ed and samples o 87
hepa opanc eas, man le, gonad, gills and ex apalial luid we e collec ed. Fo seed o 88
young clams, he whole o ganism o 15-20 indi iduals was collec ed and ea ed as he89
dissec ed o gans. Homogena es we e p epa ed by adding a olume o saline solu ion90
(0.85%) o he weighed o gan samples (1:1). Samples we e se ially dilu ed in saline91
solu ion (0.85%), pla ed on ma ine aga (MA) (P onadisa, Mad id, Spain) and92
hiosulpha e ci a e bile suc ose aga (TCBS, Oxoid L d., Basings oke, UK), and93
incuba ed a 23 ± 1ºC o 10 days (MA) o 48h (TCBS). Pu e cul u es o he di e en 94
colony mo phologies we e eco e ed on MA. Isola es we e ou inely g own on Ma ine95
Aga (P onadisa) a 23 ± 1ºC o 24 hou s. S ock cul u es we e main ained ozen a 96
–80oC in Ma ine B o h (Oxoid) supplemen ed wi h 15% glyce ol ( / ).97
Pheno ypic analysis98
The bac e ia isola ed (n = 759) we e subjec ed o a se o pheno ypic es s: cell99
mo phology and mo ili y, G am s ain, oxidase, g ow h on TCBS, suscep ibili y o he100
ib ios a ic agen 0/129, p oduc ion o a ginine dihyd olase, lysine and o ni ine101
deca boxylase, Glucose e men a ion, indole, hyd olysis o gela in, s a ch, esculin and102
Tween 80, educ ion o ni a e o ni i e, p oduc ion o gas om glucose, Voges103
5
P oskaue , Me hyl ed, g ow h a di e en empe a u es (4ºC, 37ºC, 44ºC) and a 104
di e en salini ies (0%, 0.5%, 3%, 6%, 8%). [1, 9, 24].105
Geno ypic analyses106
A o al o 145 ep esen a i e s ains o all pheno ypic g oups we e selec ed o u he 107
geno ypic analysis. A leas one s ain was selec ed o each pheno ypic g oup. The 145108
selec ed s ains o inge p in ing by AFLP included 33 isola ed om si e A, 36 om109
si e B, 29 om si e C and 47 om si e D.110
Genomic DNA ex ac ion111
A loop o bac e ial cells was ha es ed om esh cul u es o genomic DNA ex ac ion112
using Easy DNA (In i ogen, Ba celona, Spain) ki . Concen a ion and pu i y o DNA113
we e es ima ed measu ing op ical densi ies a 260 and 280 nm using a114
spec opho ome e Spec a Max Plus 384 (MDS Inc., Sunny ale, CA). DNA in eg i y115
was e i ied by loading DNA samples on a 1% aga ose gel in 1X TAE bu e (40Mm116
T is/Ace a e, 1Mm EDTA, pH 8.0). Ex ac ed DNA was main ained a –20ºC un il117
u he use.118
AFLP analysis119
AFLP is based on he ampli ica ion o subse s o genomic es ic ion agmen s by using120
PCR [11, 33]. DNA s ock samples we e dilu ed o ob ain an ini ial concen a ion o 1 µg121
o DNA in 26 µl o s e ile Milli Q wa e . DNA was diges ed wi h TaqI (5´TCGA3´) and122
HindIII (5´AAGCTT3´) (Ame sham Pha macia Bio ech, Sweden) and double-s anded123
adap o s we e liga ed o he ends o es ic ion agmen s wi h T4 ligase (Ame sham124
Pha macia Bio ech) o gene a e empla e DNA o PCR ampli ica ion. Two subsequen 125
PCR ampli ica ions, p e-selec i e PCR and selec i e PCR, we e pe o med using126
p ime s and condi ions p e iously desc ibed [25] in a GeneAmp PCR Sys em 9600127
he mocycle (Applied Biosys ems, USA). Sepa a ion o he selec i e PCR p oduc s128
6
was pe o med on a 36 cm dena u ing polyac ilamide gel (4.25% Ac ylamide, 6 M U ea129
in 1 x TBE/89 mM T is + 89 mM Bo ic acid + 2mM EDTA, pH 8.3) on an ABI P ism130
377 DNA sequence (Applied Biosys em). The le el o ep oducibili y was con olled131
by gene a ing he AFLP pa e n o he s ain V. alginoly icus LMG 4409T om he132
BCCM/LMG Bac e ia collec ion (Belgian Co-o dina e Collec ions o Mic o-133
o ganisms/Labo a o y o Mic obiology o he Ghen Uni e si y, Belgium) in each134
AFLP assay pe o med. T acking and no maliza ion o he lanes we e pe o med using135
he Gene Scan 3.1 so wa e (Apple a Co., No walk, CT), and he subsequen nume ical136
was ca ied ou by he BioNume ics 4.5 so wa e (Applied Ma hs, Sin -Ma ens-La em,137
Belgium). Simila i y alues we e calcula ed using he Dice coe icien ( ole ance alue138
o 0.3%) and a dend og am was cons uc ed using he UPGMA algo i hm. The AFLP139
pa e ns we e compa ed wi h he p o iles o he Vib ionaceae da abase a he140
BCCM/LMG Bac e ia Collec ion, con aining 544 e e ence p o iles ep esen ing mos 141
alidly desc ibed species gene a ed mainly by Thompson e al. [25]. The cu -o le el o 142
simila i y o de ining he AFLP clus e s was 63%. In addi ion, s ains showing143
simila i ies highe han 88% we e conside ed as clones ollowing he c i e ia o 144
Thompson e al. [25].145
Sequencing o he 16S RNA, poA and ecA genes146
Clus e s ha could no be iden i ied by AFLP we e u he analyzed by sequencing he147
16S RNA gene o ep esen a i e s ains as p e iously desc ibed [20]. 16S RNA genes148
we e ampli ied by PCR wi h uni e sal p ime s pA and pH [10]. P ime s [5]149
co esponding o in e nal conse ed egions o his gene we e used o he sequencing150
eac ions in Tg adien machine(Biome a, Ge many), using a GenomeLab DTCS-Quick151
S a Ki (Beckman Coul e , USA). Sequencing o he house-keeping genes poA and152
ecA was pe o med acco ding o Thompson e al. [23, 32].153
7
Sequencing p oduc s we e analysed using an ABI P ism 373A DNA sequence (Applied154
Biosys ems). Sequence da a analysis was pe o med wi h DNAs a Seqman p og am155
(Lase gene, USA). Gene sequences o he no el s ains we e subjec ed o a BLAST156
sea ch agains he la es elease o he GenBank and ela ed sequences we e ob ained.157
Phylogene ic ees we e cons uc ed by neighbo -joining (NJ), dis ance ma ices we e158
calcula ed using Kimu a´s wo pa ame e co ec ion and s abili y o g oupings and159
boo s ap analysis (1000 eplica es) was pe o med wi h he p og am Mega e sion 3.1.160
161
RESULTS AND DISCUSSION162
A o al o 759 s ains we e ob ained om cul u ed clams, assigned o he Genus Vib io163
on he basis o 25 mo phological, physiological and biochemical cha ac e is ics, and164
g ouped in o 29 dis inc clus e s (Table 1). V. splendidus u ned ou o be ep esen ed by165
428 isola es i.e. 56% o he o al numbe o isola es. The nex mos nume ous g oups166
we e V. alginoly icus (48 isola es, 6.3%), V. diazo ophicus (41 isola es, 5.4%), V.167
aes ua ianus (32 isola es, 4.2%), V. pelagius I / V. supe s es (31 isola es, 4.1%) and V.168
lu ialis (28 isola es, 3.7%). The es o he g oups con ained less han 20 isola es. Some169
g oups we e qui e homogeneous, consis ing o isola es ha ing almos iden ical170
p ope ies; o he s we e somewha he e ogeneous including isola es wi h a iable171
pheno ypic ai s. Recen ly, Nogue ola and Blanch [18] ha e p oposed a se o 172
dicho omous keys o apid iden i ica ion o Vib io isola es. Wi h mino excep ions, ou 173
esul s we e in ag eemen wi h he iden i ica ion schemes po posed in hei wo k.174
Disc epancies we e obse ed o some ai s in ew species, such as g ow h a di e een 175
empe a u es and salini ies o Voges-P oskaue es in V. diazo ophicus, V. scoph halmi176
and V. na iengens. These disc epancies we e p obably due o he ac ha hose au ho s177
8
s udied only he ype s ains o he di e en species ins ead o a la ge numbe o 178
en i onmen al isola es as included in ou wo k.179
The AFLP analysis was pe o med on 145 ep esen a i es o he di e en pheno ypical180
g oups and included also ype and e e ence s ains o p ac ically all known species o 181
he genus Vib io. The AFLP pa e ns con ained om 65 o 156 bands be ween 50 o 536182
bp in size. No clonali y was obse ed in any o he clus e s, being he AFLP simila i y183
alues among s ains always unde 88%. The simila i y among pa e ns ob ained o V.184
alginoly icus LMG 4409T employed as posi i e con ol in e e y AFLP un was always185
highe han 88%, suppo ing he meaning o his alue as cu -o o clonali y.186
A o al o 94 clus e s and 81 unclus e ed s ains we e ob ained (Dend og am is a ailable187
as Supplemen a y File). A o al o 57 Galician isola es we e iden i ied o species le el188
and dis ibu ed in 13 clus e s. Species iden i ied we e V. cycli ophicus (17 s ains), V.189
splendidus (16 s ains in wo clus e s), V. alginoly icus (5 s ains), V. diabolicus (4190
s ains), V. c assos eae (4 s ains), V. chagasii (3 s ains), V. medi e anei (2 s ains),191
V. ich hyoen e i (1 s ain), V. pa ahaemoly icus (1 s ain), V. pec enicida (1 s ain) and192
V. len us (1 s ain).193
Mos clam s ains (n = 88) emained uniden i ied by AFLP, since hey did no clus e ed194
wi h any ype s ain. Fi y nine o hem we e dis ibu ed o e 16 clus e s, while 29 we e195
unclus e ed. The clus e s ob ained in he p esen wo k we e simila o hose ob ained by196
Thompson e al. [25] using he DICE coe icien and he WARD algo i hm, al hough an197
impo an imp o emen o he me hod o iden i ica ion pu poses was achie ed, since198
no g oups ha bo ing mul iple ype s ains we e obse ed. On he o he hand, some o 199
he species analyzed including V. splendidus, V. ha eyi o V. diazo ophicus, g ouped200
in mo e han one clus e indica ing he exis ence o in aspeci ic di e si y wi hin hese201
9
Vib io species. Simila esul s ha e been p e iously obse ed by o he au ho s using202
his echnique o yping o di e en Vib io species [13, 28].203
A wide geog aphical dis ibu ion was obse ed o mos pheno ypic and AFLP g oups204
es ablished, being no possible any associa ion among g oups and a speci ic si e. The205
highes di e si y was obse ed in si e D, bu his ac is p obably ela ed o he highe 206
numbe o s ains (33%) ob ained om ha si e.207
A compa ison o he pheno ypic and he AFLP esul s showed ha , o isola es ha 208
could be iden i ied a species le el by bo h p ocedu es, iden i ica ion was coinciden 209
only in 29.82% o he cases. Majo disc epancies we e obse ed in he he e ogeneous210
species, such as V. splendidus [8]. Thus, some isola es conside ed as V. splendidus211
based on pheno ypic esul s, we e iden i ied as V. cycli ophicus, V. c assos eae, V.212
chagasii o we e uniden i ied by AFLP.213
Sequencing o he 16S RNA gene o ep esen a i e s ains belonging o he majo 8214
uniden i ied clus e s, con i med ha hese isola es belonged o he genus Vib io. Clus e 215
5 (6 s ains) could be assigned o V. asmaniensis (99.74% simila i y). Clus e s 37 (7216
s ains), 48 (3 s ains) and 86 (4 s ains) may cons i u e h ee po en ial new species217
aken in o accoun hei 16S RNA, poA and ecA simila i ies (Table 2), as well as218
hei ela i e posi ions o known ib ios in he phylogene ic conca ena ed ee (Fig. 2).219
These h ee po en ial new species a e pending o u he analysis o sa is y cu en 220
equi emen s o new species desc ip ions [22]. Clus e 37 was close o he V.221
halio icoli-like g oup showing sequence simila i ies in he 16S RNA gene o 99.4%222
wi h Vib io comi ans, 99.15% wi h Vib io a us and 99.0% wi h Vib io inusi a us223
(Table 2). Howe e , poA and ecA simila i ies we e lowe han 97 and 93%224
espec i ely (Table 2), and DNA-DNA hyb idiza ion alues ob ained among hese225
species did no each 36% (da a no shown). Clus e 48 was close o he V. splendidus-226
16
395
[33] P. Vos, R. Hoge s, M. Bleeke , M. Reijans, T. Van de Lee, M. Ho nes, A. F ij e s,396
J. Po , J. Peleman, M. Kuipe , M. Zabeau. AFLP: a new echnique o DNA397
inge p in ing. Nucleic Acids Res. 23 (1995) 4407-4414.398
399
[34] M. Waech e , F. Le Roux, J.L. Nicolas, E. Ma issal, F. Be he. Cha ac e iza ion o 400
pa hogenic bac e ia o he cupped oys e C assos ea gigas. C. R. Biol. 325 (2002)401
231-238.402
17
Table 1. Da a o p elimina y pheno ypic da a and AFLP iden i ica ion clus e s. NT, no es ed.
RP= Rudi apes philippina um, RD= Rudi apes decussa us
Pheno ypic
g oup
AFLP clus e s
Pheno ypic iden i ica ion
AFLP iden i ica ion
Geog aphic si es
P1
5, 6, 7, 9, 13, 19,
48, 68, 77, 26
V. splendidus I
V. splendidus, V. chagasii,
V. cycli ophicus, uniden i ied
A, B, C, DRP, DRD
P2
5, 7, 8, 9, 13, 16,
19, 48
V. splendidus II
V. splendius, V. c assos eae,
V. chagasii, V. cycli ophicus, uniden i ied
A, B, C, DRP, DRD
P3
28, 29
V. alginoly icus
V. alginoly icus, V. diabolicus
A, B, C, DRP, DRD
P4
7, 8, 19, 68, 70
V. diazo ophicus
V. splendidus, V. c assos eae,
V. cycli ophicus, uniden i ied
A, B, C, DRP, DRD
P5
5, 14, 19, 29
V. aes ua ianus
V. len us, V. cycli ophicus, V. diabolicus,
uniden i ied
A, B, C, DRP
P6
36, 37, 53, 67, 70
V. pelagius I / V. supe s es
Uniden i ied
A, B, C, DRP, DRD
P7
6, 7, 9, 16, 26
V. lu ialis
V. splendidus, uniden i ied
A, B, C, DRP, DRD
P8
5, 37
V. na iengens
Uniden i ied
A, B, C, DRP
P9
28, 53
Vib io sp.
V. alginoly icus, uniden i ied
A, B, C, DRP, DRD
P10
9, 19, 26, 70, 82
V. pacinii
V. cycli ophicus, uniden i ied
A, B, C, DRP, DRD
P11
13, 16, 19
V. len us
V. chagasii, uniden i ied
A, B, C, DRP, DRD
P12
22, 48, 70
V. asmaniensis / V. o is
V. ape is, uniden i ied
A, B, C, DRD
P13
28, 58
V. ische i / V. logei
V. alginoly icus, uniden i ied.
A, B, C, DRD
P14
70, 82
V. scoph halmi
Uniden i ied
A, B, C, DRP, DRD
P15
8, 19
V. cycli ophicus
V. cycli ophicus, V. c assos eae
A, B, C, DRP, DRD
P16
52
V. o dalii
V. ich hyoen e i
B, C
P17
8, 29, 47, 55
V. ha eyi / V. ulni icus
V. c assos eae, V. diabolicus,
V. pa ahaemoly icus, V. medi e anei
A, B, C
P18
19, 24
V. ne eis
V. cycli ophicus, V. pec enicida
A, B, C, DRP
P19
19, 77
V. my ili
V. cycli ophicus, uniden i ied
B, DRD
P20
22
V. ape is
V. ape is
A, DRP
P21
19, 69, 82
V. pelagius II
V. cycli ophicus, uniden i ied
B, DRD
P22
37
V. cicinnan iensis
Uniden i ied
B, DRP, DRD
P23
37
V. pec enicida
Uniden i ied
B, DRP
P24
6, 47
V. medi e anei
V. medi e anei, V. splendidus
A, C, DRP
P25
NT
V. u nisii
NT
B
P26
NT
V. gazogenes
NT
C
P27
Unclus e ed
V. gallicus
Uniden i ied
DRD
P28
Unclus e ed
V. aga i o ans
Uniden i ied
A
P29
9
V. wodanis
Uniden i ied
B
18
Table 2. Sequence simila i ies (%) o he genes 16S RNA, ecA and poA o h ee
po en ial new Vib io species and closely ela ed species.
Species
R33677 (Clus e 37)
16S ecA poA
R33624 (Clus e 48)
16S ecA poA
R33755 (Clus e 86)
16S ecA poA
V. comi ans
V. a us
V. inusi a us
V.splendidus
V. gigan is
V. pome oyi
A. wodanis
A. ische i
A. logei
A. salmonicida
99.4 93.0 99.8
99.1 84.0 94.0
99.0 91.0 96.0
- - -
- - -
- - -
- - -
- - -
- - -
- - -
- - -
- - -
- - -
97.6 86.5 97.8
97.3 86.0 97.0
97.1 86.0 97.8
- - -
- - -
- - -
- - -
- - -
- - -
- - -
- - -
- - -
- - -
98.1 82.0 98.1
97.1 89.0 97.2
97.8 82.0 97.4
96.0 81.0 97.5
19
Table 3. Accession numbe s o sequences o he genes 16S, ecA and poA de e mined
in he labo a o y.
S ain
Clus e
16S
ecA
poA
R33660
R33635
R33634
R33656
R33677
R33624
R33727
R33755
V. gigan is DSM 18531T
V. c assos eae LMG 22240T
V. comi ans LMG23416T
V. a us LMG 23674T
V. inusi a us LMG 23434T
5
9
16
26
37
48
70
86
-
-
-
-
-
EF599163
EF500162
EU541607
EU541606
EF599161
EU541605
EF599164
EU541604
-
-
-
-
-
EU541589
EU541590
EU541592
EU541591
EU541585
EU541587
RU541588
EU541586
EU541593
EU541594
EU541597
EU541599
EY541600
EU541569
EU541570
EU541572
EU541571
EU541565
EU541566
EU541568
EU541567
EU541573
EU541574
EU541577
EU541578
EU541579
20
Figu e legends
Fig. 1.- Sampling si es in he Galician coas (NW Spain).
Fig. 2.- NJ Phylogene ic ee based on conca ena ed sequences o h ee genes ecA,
poA, and he 16S RNA gene. Ho izon al b anch leng hs a e p opo ional o
e olu iona y di e gence. Boo s ap pe cen ages om 1000 eplica es appea nex o he
co esponding b anch. Simila esul s we e ob ained using he Maximum-Pa simony
me hod (da a no shown).
21
Fig. 1.- Beaz-Hidalgo e al.
D
A
C
B
22
Fig. 2. Beaz- Hidalgo e al.
V. u nisii ATCC 35016T
V. lu ialis ATCC 33809T
V. p o eoly icus ATCC 15338T
V. chole ae ATCC 14035T
V. cincinna iensis ATCC 35912T
V. gazogenes ATCC 29988T
V. penaeicida LMG19663T
V. ubiashii ATCC 19109T
V. ne eis ATCC 25917T
V. na iegens ATCC14048T
V. alginoly icus ATCC 17749T
V. campbelli ATCC 25920T
V. ha eyi ATCC 14126T (X74706.1)
V. aginoly icus g oup
V. pec enicida A365 (Y13830.1)
V. scoph halmi CECT 4638T (VSU46579)
V. ich hyoen e i LMG 19664T (AJ437192.1)
V. aes ua ianus ATCC 35048T (X74689.1)
V. o dalii ATCC 33509T (X74718.1)
V. medi e anei CIP 103203T (X74710.1)
V. pelagius ATCC 25916T (X74722.1)
V. chagasii LMG 13237 (AJ490157.1)
R33624 (Clus e 48)
V. cycli ophicus LMG 21359T (AM162656.1)
R33634 (Clus e 16)
V. len us CECT 5110T (AJ278881.1)
R33660 (Clus e 5)
V. asmaniensis LMG 21574T (AJ514912.1)
V.splendidus ATCC 33125T (X74724.1)
V. kanaloae LMG 20539T (AM162657.1)
R33635 (Clus e 9)
R33727 (Clus e 70)
V. c assos eae CAIM 1405T (EF094887.1)
R33656 (Clus e 26)
V. gigan is CAIM 25T (EF094888.1)
V. pome oyi LMG 20537T (AJ491290.1)
V.comi ans LMG 23416T (DQ922915)
V.inusi a us LMG 23434T (DQ922920)
R33677 (Clus e 37)
V. halio icoli IAM14596T (AB000390)
V. neona us LMG 19972T (AY426979)
V. ezu ae LMG 19970T (AY426980)
V. umoiensis LMG 20038T (AB013297.1)
R33755 (Clus e 86)
A. ishe i ATCC 7744T (X74702.1)
A. wodanis NVI 88/441T (AJ132227.1)
A. salmonicida NCMB 2262T (X70643)
A. logei NCIMB 2252T (AJ437616.1)
100
100
100
100
94
100
100
100
100
94
100
100
100
100
63
43
59
93
65
97
60
38
52
25
28
36
63
25
22
100
100
93
84
92
46
39
73
61
100
44
37
41
57
30
0.01
A. ische i g oup
V. halio icoli g oup
V. splendidus g oup
