Scientific Reports of the
Hokkaido Salmon HatcheryNo 48: 23-29, lLIarch 1994

Existence of Non-Agglutinating Aeromonas salmonicida subsp.
sazmonicida in Strains Isolated from Salmonids
in Yamagata Prefecture, Japan*‘
Tetsuichi Nomura*’, Mamoru Y o ~ h i r n i z u * ~Shouzo,
M ~ k i * and~ , Yoshio Ezura*3*’ Research Division, Hokkaido Salmon Hatchery, Fisheries Agency of Japan,
2-2 Nakanoshima, Toyohira-ku, Sapporo 062, Japan*3

Microbiology Laboratory, Faculty of Fisheries, Hokkaido University,
Minato-machi 3, Hakodate, Hokkaido 042, Japan*‘ Yamagata Prefectural Inland Fisheries Experimental Station,
1-4-12 Izumi-machi, Yonezawa, Yamagata 992, Japan

Abstract.-Auto-agglutination, protein staining of colonies and protein pattern of A-layer in thirty
three isolates of Aeromonas salmonicida from salmonids in various parts of Yamagata Prefecture andHokkaido were studied. The strains isolated in Yamagata Prefecture did not showed auto-agglutinationin broth, colony colour was mainly grey-white on TSA-C and NA-C medium, and there was no 50
kDa. major protein hand in the A-layer with SDS-PAGE. It was revealed from the results that
non-agglutinating A. salmonicida exist in the strains isolated in Yamagata Prefecture. While there wereno strains isolated in Hokkaido which were non-agglutinating. We suspect that the reason for the
existence of non-agglutinating strains in Yamagata Prefecture may depend on the temperature at which
this bacterium was isolated.

Introduction

Materials and Methods

Auto-agglutination of Aeromonas salmonicida, causative
agent of fish furunculosis, has assumed the importantcharacter of pathogenicity. After the first detailed electron
microscopic studies of Udey and Fryer (1978), many
reports indicated that strong auto-agglutinatingproperty of
A. salmonicida was found to correspond with virulence andthe presence of A-layer on the surface of the agent (Munnet al., 1982; Phipps et al., 1983; Austin and Austin, 1987;
Ellis et al., 1988; Bernoth, 1990).
In general it was believed that all fresh isolates of A.
salmonicida from diseased fish have auto-agglutinating
properties (McCarthy and Rawle, 1975; Kirnura andYoshimizu, 1984; Sakai, 1985; Sakai and Kimura, 1985;
Udey and Fryer, 1978).
This study attempted to show that non-agglutinating A.
salmonicida strains exist and can be isolated from diseasedfish in Yamagata Prefecture, Japan.

Bacterial strains. Thirty three A. salmonicida strainsused in this study were isolated from kidneys of diseased
chum salmon (Oncorhynchus keta), pink salmon ( 0 .
gorbuscha), sockeye salmon ( 0 . nerka), masu salmon ( 0 .
masou), Japanese char (Salvelinus leucomaenis) andbrook trout ( S . fontmalis) in various parts of Yamagata
Prefecture and Hokkaido. The date, place of isolation, andhost species are shown in Table 1. A. salmonicida wasisolated from fish at 25°C and 20°C in Yamagata Prefecture and Hokkaido, respectively. The isolates weremaintained on nutrient agar (NA; Eiken Chemical Co.,Tokyo) slants at 20°C until used for this study.
Culture media. Nutrient broth (NB), torypto-soya
broth (TSB) and torypto-soya agar (TSA) were purchased from Eiken Chemical Co. (Tokyo).

Contribution A No. 343 from the Hokkaido Salmon Hatchery*‘Epidemiological studies of furunculosis in salmon propagation-IV-

Examination of biochemical characteristics.
Biochemical characteristics of each strain were examinedat 20°C by the methods of Cowan (1974).
Auto-agglutination. Auto-agglutination of thestrains isolated in Yamagata Prefecture were determined
one week after isolation from fish and the strains isolatedin Hokkaido were determined from one to ten years after.

23 -

.

3 M 5 ?&@@SCI REP HOKKAIDO SALMON HATCHERY,NO. 48, 1994

Auto-agglutination was determined by the relative rates ofsedimentation of bacteria in NB at 20°C over 48 hours.
Auto-agglutinating properties were assigned as follows;
cells which gathered at the bottom of the culture tube witha clear supernatant were strong auto-agglutinating (+strains. The strains whose some of the cells sank to thebottom of the tube and the supernatant was cloudy wereweak auto-agglutinating (+) strains. The strains whose
the cells did not sink to the bottom of the tube were a
non-agglutinating (-) strains.
Protein staining of colonies. Subcultures of eachstrain were prepared on NA and TSA, supplemented with
0.1 mg/ml Coomassie brilliant blue R250 (Sigma Chemi-+)

cal Co., St. Louis, U.S.A) according to the method of
Evenberg et al. (1985). This medium was referred to as
NA-C and TSA-C respectively. Plates were incubated at
2 0 C for 3 days. The protein staining of strains according
to the colour of the colonies obtained by growth on NA-C
or TSA-C was as follows; colonies which were grey-whitein colour was classified as a negative strain, whereascolonies with a dark blue and a faint halo around the colony
was a positive strain.
Extraction of A-layer. Extraction of A-layer wasdone by the method of Ward et al. (1985). After growth in
TSB for 24 hours at 20"C, cells were harvested, andwashed three times in PBS (phosphate buffer saline, pH

Table 1. Source of Aeromonas salmonicidu.
StrainHost species

Strains isolated in Yamagata
YM- 1
Chum salmon
YM- 2
Chum salmon
Chum salmon
YM- 3
YM- 4
Chum salmon
YM- 5
Chum salmon
YM. 6
Chum salmon
YM- 7
Chum salmon
Chum salmon
YM- 8
YAl- 9Chum salmon
YM-10
Japanese charI'M-12
Japanese char
YM-13
Japanese char
YM-14
Japanese char
YM-15
Japanese char
YM-16
Japanese char
YM-17
Masu salmon
YM-20
Masu salmon
YM-22
Masu salmon
YM-23
Brook trout
YM-24
Brook trout
YM-25
Brook trout
YM-27
Japanese char
YM-28
Japanese char
Strains isolated in Hokkaido*'
I IK-20- 1
Chum salmon
HK-KO~lSockeye salmon
HK-9037
Chum salmon
HK-90111
Chum salmon
HK-9008
Chum salmon
HK-90141
Chum salmon
HK-90146
Chum salmon
HK-9201
Masu salmon
HK-9202
Masu salmon
IIK-9301
Pink salmon

Place

Date of Isolation

Tsuruoka City
Tsuruoka City
Tsuruoka City
Tsuruoka City
Tsuruoka City
Tsuruoka City
Tsuruoka City
Tsuruoka City
Tsuruoka City
Obanazawa City
Obanazawa City
Obanazawa City
Obanazawa City
Obanazawa City
Obanazawa City
Iitomi Chou
Iitomi Chou
Iitomi Chou
Yahata Chou
Yahata Chou
Yahata Chou
Yamagata City
Yamagata City

March 24, 1991
March 24, 7991
March 24, 1991
March 24, 1991
March 24, 1991
March 24, 1991
March 24, 1991
March 24, 1991
March 24, 1991
March 8, 1991
March 8, 1991
March 8, 1991
June 21, 1991
June 21, 1991
June 21, 1991
July 16, 1991
July 16, 1991
July 16, 1991
July 29, 1991
July 29, 1991
July 29, 1991
Sept. 24, 1991
Sept. 24, 1991

Tokachi River
Chitose Branch
Shibetsu River
Tokoro River
Shibetsu Kiver

Sept. 14, 1980
Jan. 10, 1989
Sept. 21. 1990
Oct. 2, 1990
Sept. 21, 1990

Ishikari Kiver

Oct. 11, 1990

Shikyuu River
Yakumo Hatchery
Yakumo Hatchery
Abashiri River

Oct. 25, 1990
July 20, 1992
July 20, 1992
Aug. 20, 1993*' Nomura et al. (1993)- 24 -NOMURA ET AL. -NON-AGGLUTINATING A. SALMONICIDA

7.5). After washing, A-layer protein was extracted fromthe cells by suspending them for 15 min. at room temperature in aqueous 2 M guanidine hydrochloride (Tokyo
Kasei Co., Tokyo), the insoluble material was removed bycentrifugation. A-layer was precipitated from the clear
supernatant by dialysis against distilled water and thispartially purified A-layer was used in analysis of protein
Table 2. Characteristics of the strains used in thisstudy and Aeromonas salmonicida subsp. salmonicida (Austine and Austine, 1987)
Strains used A. salmonicida
in thissubsp.
study
salmonicida

Characteristics
Gram stain
Motility
Growth in at 37°C
Brown, diffusible pigment
OxidaseO/F test
Esculin hydrolysis
Arginine dihydrolase
Lysin decarboxylase
Ornithine decarboxylase
Citrate utilization
H,S from cysteine
UreaseIndole production
Voges- Proskauer
Geltin hydrolysis
Growth in KCN brothProtease
Growth in 1% NaCl
2% NaCl
3% NaCl
4% NaCl
5% NaCl
Fermentation of:
GlucoseMannitolInositol
SorboseRhamnose
SucroseRaffinose
MaltoseMannoseFructose
LactoseArabinose

+
+
F
+

+
+

-

V
V

-

-

F

t

Results
Biological c h a r a c t e r i s t i c s . Biological characteristics of the strains used in this study are shown in Table
2. The strains showed the same characteristics. Gram
negative, non-motile, cytochrome oxidase positive, no
growth at 3 T C , production of a diffusible brown pigment.
Comparison of the strain characteristics and the reports of
A. salmonicida subsp. salmonicida by Austin and Austin(1987), showed that all the strains used in this study were
A. salmonicida subsp. salmonicida
A u t o - a g g l u t i n a t i o n of the s t r a i n s . Autoagglutinating properties of the strains are showed in Table
3. YM-1, YM-7, YM-17 and YM-24 strains were weakauto-agglutinating (+) strains, and 10 strains isolated in
Hokkaido were strong auto-agglutinating+) strains,while, other strains did not show the auto-agglutinating
property in NB at 20°C and were designated as nonagglutinating (-) strains.
Protein staining of colonies. The colour of thecolonies obtained by growth on NA-C and TSA-C, differentiated the isolates into negative (Fig. 1 A) and positive
strains (Fig. 1B). The four strains isolated in Yamagata
Prefecture were to negative strains but the colony colour
was mainly grey-white with dark blue colonies (-/+:
Table 3). While the other 19 strains showed only greywhite colonies and were differentiated as negative strains.
All the 10 strains isolated in Hokkaido were positive
strains.
Protein pattern of A-layer on S D S - P A G E . The
electrophoretically analyzed protein pattern of A-layerfrom YM-2 and HK-20-1 are shown in Fig. 2. The SDSP A G E showed that an A-layer extracted from the autoagglutinating strain, HK-20-1, possessed a major protein
which was not present in the non-agglutinating strain,
YM-2. It was revealed that this additional protein of
HK-20-1 had a mol. wt. of 50 kDa. (Fig. 3).
The existence of the 50 kDa. mol. wt. band by SDSP A G E in the strains examined is shown in Table 3. Thisband was existed in the A-layer extracted from the strong

(+

-

t

-

+

-

+
+
+
+

V

+

t

+

+
+

+

-

+

t ,positive reaction; -, indicates negative reaction;
F, fermentative; , not done; V,variable result.

-pattern with sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE).
SDS-PAGE. The protein pattern of the partially purified A-layer was analyzed with SDS-PAGE. SDS-PAGE
was carried out using ATO-system (At0 Scientific Co.,Tokyo, Japan) with 12.5 % separate gel, 4% stacking gel
and the buffer system of Laetnmli (1970). The L M W
electrophoresis calibration kit for SDS-PAGE (Pharmacia Fine Chemical, Uppsala, Sweden) was used forstandard proteins to estimate molecular weight.

- 25 -

S h .1T&WOgSCI REP HOKKAlDO SALMON HAKHERY, NO 48.1994
auto-agglutmation strains isolated m H o h d o . Although,the A-layer from the "on-agglutmating s t r m s isolated m
Yamagala Prefecture did no1 showed the emstcnee 01 thisband. The A-layer from the we& auto-agglutmatmg
strains, YM-1. YM-7, YM-17 and YM-24. showed u i s tenee of hmt band of 50 D a . pmtem.

Discussion
There are many reports about auto-aggluUnating properties of A. salmameida subsp. sohnonrcdo. the cpusattlve
agent of fish furuneulosls. m e e the first report by Udey
and Fryer (1978). Recently. relationships between vimlence and the existence of A-layer on the surface of h ebacterium have been presented m several reports (Ishlguroe l al., 1981; Kay ct d.,1981; Ward et al., 1985)
In general I t has been suspected that dl of s t r m s of A.
s o h l n d o isolated from diseased fish have autonggluhnating pmperties at the Lime of isolatlan. McCarlhy
and Rawle (1975) , Udey and Fryer (1978) reported thatthe strams isolated m the United Slates and Bntish
Columbia, Canada, showed strong auto-aggluhnatmgpropertles at lhe Llmc of Isohhon.
Saka (1985) showed that 100 % of the A solmonlczdo
strams Isolated m Hokkaido possessed auto-%glutmationfor 2 years after aalation, and Kimura and Yashi-u(1984) also reported that 215 strains isolated fromchumsalmon. pink salmon and mas" salmon which showed noapparent clinical signs of huruneulosls had autoagglutlnatlng properhcs. However these reports concerned
the isolates in Hokkado. As far as d IS known, there IS noreports 01 a auto-aggluhnatlan tm isolates fmm the Honshuma.
Recent unconfirmed reports have been made of a strun
wbich does no1 show auto-agglutloahon eharactenstles
from the Japanese char m Honshu (Monkma, personalcommuniuhon). In this study, we compared autaagglutlnahng properties by broth, colony eolaur on solid
medium supplemented with Coomassic brilliant blue. andouter membrane pmtem pattern by SDS-PAGE, of stransisolated from Ymagata and Hokkada Prefectures. J a p .
We showed that A. solmmundo s t r m s whleh dld no1 show
auta-agghIhnation m culture medium, posihve proternstaining of colonies with Coomzssie bnlhant blue or P
major protein with molecular weight 50 ma..
were presentm the strams isolated in Y m a g a k Prefecture.
Bcmoth (1990) presented thal TSA-C is suitable as ascreening medium for the presence of A-layer and hurthermore, aub-agglutination m distilled water always c o m lates with A-lay-. In this report. we observed that aumaggluunation of the s t r m s used m lhis study eomktees

Fig. 1. Protein staining of colonies on nutrient agar
supplemented with 0 ~ 1rnglrnl Caornassie brilliant
blue R-250~A. greywhite colour colonies fromnegative strain. YM-2. B, dark blue colour colonies
from positive stram, HK-20-1
mth protein staining of colonies and A-layer.
Ward et al. (1985) indicated lhat guanidlne extracts ofauto-%glutmating A. salmonlrda stram display a band
eomspondlng to 52 kDa. and lhe extracts of nan%glutmaling strain did not contain the band. Also, thestrams which did not showed auto-agglulmaling property
m his report did not have the major protein with molcculir
wunght o f 50 kDa. whereas this protein was present m 111
auto-agglutmattmg strams isolated in Hokkuda.
Fram the results of this reporl it IS obvious that all thestran isolated from Yunagata were non-agglutmating A.
salmonicida strams.

The reason why the strains isolated in Yarnagala Prefecture did not show auto-agglulinatmn in NB IS unknown.
The property of aum-agglulinahon is lost when the strain
IS cultured for long tune m culture medium, and It 1s
possible lhat the property of the s t r m s used m this study
might have changed smcc their ongmal sola ah on.

26

NOMURA ET AL. -NON-AGGLUTINATING A. SALMONICIDA

Table 3. Auto-agglutination in nutrient broth, colour of the colonies obtained by growth on
nutrient agar (NA-C) and trypto-soya agar (TSA-C) supplemented with 0.1 mg/rnl Coomassie brilliant blue R-250 and exist of protein with m.w. 50 kDa. in A-layer.
StrainAutoagglutination*'

Strains isolated in Yamagata
YM-1
YM- 2
YM- 3
-

+

YM- 4
YM- 5
YM- 6
YRII- 7
YM- 8
YM- 9
YM.10
YM-12
YM-13
YM-14
YM-15
YWI-16
YM-17
YM-20
YM-22

NA-C*2

TSA-C*3

50 kDa. protein*'

-/+

-/+

tr

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-/+

-/+

tr

~

~

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

-/+

-/+

tr

-

-

-

-

-

-

-

-

-

-

-

-

+

-/+

-/+

tr

-

-

-

-

-

-

-

-

-

-

-

-

Strains isolated in Hokkaido
HK-20-1
HK-KO-1
HK-9037
HK-90111
HK-9008
HK-90141
HK-90146
HK-9201
HK-9202
HK-9301

+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+

+

YM-23
YM-24
YM-25
YM-27
YM-28

++
++
++
++

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

+
+
+
+
+
+
+
+
+

+ +,

Auto-agglutination: -, non-agglutinating strain; +, week auto-agglutinating strain;strong auto-agglutinating strain.
* 2 NA-C: +, dark blue colonies; -,grey-white colonies; -/+, mainly grey-white colonies
with dark blue colonies.
* 3 TSA-C: same as NA-C.
*' 50 kDa. protein in A-layer: -, not present; +, present; tr, present in trace amount.

*I

-

27 -

$ I+

- T~&'+fl

SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 48, 1994

5

1

2

3

a
b
C

d

1

I
I

I

0

e

2

4

Migrate Distance (cm)fFig. 2. JDS-PAGE of A-layer extracted from Aerornonas salmonicida. Lane 1, molecular weightstandards (a, 94 kDa ; b, 67 kDa ; c, 43 kDa.; d, 30
kDa.; e, 20 kDa.; f, 14.4 kDa.); Lane 2, A-layer

extracted from auto-agglutinating strain, HK-20-1;
Lane 3, A-layer extracted from non-agglutinatingstrain, YM-2.

Fig. 3. Molecular weight determination of the major
protein in A-layer of HK-20-1showed in Fig. 2 withcalibration curve established using the LMW
electrophoresis calibration kit for SDS-PAGE(Pharmacia Fine Chemical, Uppsala, Sweden) on
a 12.5% gel. 0 , the major protein band in A-layer
extracted from auto-agglutinating strain, HK-20-1;
0,the proteins in the LMW electrophoresis calibration kit for SDS-PAGE.
culture from the fish. Further study may reveal relationships between culture temperature at isolation and existence of non-agglutinating strains of A. sulmonicidu.

Acknowledgments
Sakai (1985) has reported that auto-agglutinating properties did not change for 2 years through culture. Also inthis report, we used the strains isolated in Hokkaido andthese were cultured from one to 11 years until them use inthis study, but these were strong auto-agglutinatingstrains. We determined the properties of the strains from
Yamagata one week after isolation and we do not consider
that the strains which were non-agglutinating had lost thisability in culture medium at least in the short term.
The incubation temperature was different between
strains from Yamagata and Hokkaido. Ishiguro et al.
(1981) reported that growth temperature above 22°C is
selective for colonies lacking A-layer. The strains isolatedin Yamagata Prefecture were cultured at 25'C at the timeof isolation from the fish, thus it was suspected that thestrains lost the auto-agglutinating property in the primary

The authors gratefully thank Dr. D. W. Bruno, theScottish Office Aquaculture and Fisheries Department,
Marine Laboratory, Aberdeen, U. K. for suggestions to themanuscript. We wish to express our gratitude to Mr.
Masao Shimomura, Director of Hokkaido salmon Hatchery, and Dr. Osamu Hiroi, Chief of Research Division, fortheir interest and encouragement during the investigation.
We also acknowledges the technical assistance of Miss
Kanako Akazawa.

References
Austin, B., and D. A. Austin (1987): Aeromonads. In
Bacterial Fish Pathogens: Disease in Farmed and Wild
Fish. Ellis Horwood Limited, Chichester. pp. 111-195.
Bernoth, E. -M. (1990): Autoagglutination, growth on
tryptone-soy-Coomassie-agar, outer membrane protein

28 -NOMURApatterns

andvirulenceET

0f AeromonasAL.

‑ NON‑AGGLUTINATING

salmonicida

strains.J. Fish Diseases, 13, 145‑155.
COwan, S. T. (1974): COwan & Steel'smanual for theidentification 0f medical bacteria,

2nded. Cambridge

University Press, Cambridge (translated in Japanese by
R. Sakazaki, Kindai‑shuppan, TOky0, 1974). 335 p.
Ellis,A. E., A. S. Burrows, and K. J. Stapleton (1988):
Lack 0f relationship between virulence Of Aeromonas
salmonicida and the putative virulence factors: A‑1ayer,

extracellular proteases and extracellular haemolysins. J.
Fish Diseases, 11, 309‑323.

Evenberg, D., R. Versluis, and B. Lugtenberg (1985):
Biochemical and immunological characterization 0f thecell surface of the fish pathogenic bacterium Aeromonas

A. SALMONICIDA

epidemiological study offurunculosisIn salmon propaga‑tion in Japanese rivers. Fish. Res., 17, 137‑146.
Phipps, B. M., T. J. Trust, E. E. Ishigur0, and W. W. Kay(1983): Purification and characterization 0f the cellsurface virulent A protein from Aeromonas salmonicida.

Biochemistry, 22, 2934‑2939.
Sakai, D. K. (1985): Phenotypic variation in haemagg‑
1utinationcorresponding with spontaneous agglutinationin Aeromonas

salmonicida strains. Fish Patho1., 20, 1‑7.

Sakai, D. K., and T. Kimura (1985): Relationshipbetween
agglutinative properties 0f Aeromonas salmonicida
strains isolated from fish In Japan and their resistance tomechanisms of host defense. Fish Patho1., 20, 9‑21.

Udey, L. R., and J. L. Fryer (1978): Immunization offish

salmonicida. Biochim. Biophys. Ada, 815, 233‑244.
1shigur0, E. E., W. W. Kay, T. Ainsworth, J. B. Chamber‑
1ain,R. A. Austen, J. T. Buckley, and T. J. Trust(1981): LOss 0f virulence during culture0f Aeromonas
salmonicida at high temperature.J. Bad., 148, 333‑340.

Kay, W. W., J. T. Buckley, E. E. Ishigur0, B. M. Phipps,
J. P. L. MOnette, and T. J. Trust (1981): Purificationand disposition 0f a surface protein associated withvirulence 0f Aeromonas salmonicida. J. Bact., 147,

with bacterins 0f Aeromonas

Kimura, T., and M. YOshimizu (1984): COaggludnation
test with antibody‑sensitizedStaphylococci for rapid
serological identification Of rough strains0f Aeromonas
salmonicida. Bul1.Jap. SOc. Sci.Fish.,50, 439‑442. (InJapanese with English summary.)
Laemmli, U. K. (1970): Cleavage 0f structuralproteinsduring the assembly 0f the head of bacteriophage T4.
Nature, 227, 680‑685.
McCarthy, D. H., and C. T. Rawle (1975): The rapid
serological diagnosis 0f fish furunculosis caused by'smo0th' and 'rough' strains Of Aeromonas salmonicida.
J. gen. Microbio1., 86, 185‑187.
Munn, C. B., E. E. Ishigur0, W. W. Kay, and T. J. Trust(1982): ROle 0f surface components in serum resistanceOf virulent Aeromonas salmonicida. Infect.Immunity, 36,
1069‑1075.
NOmura, T., M. YOshimizu, and T. Kimura (1993): An

― 29

Fish.

Autoaggregation 0f virulent Aeromonas salmonicida
strains lacking additional surface layer. f@ Fish and

ShellfishPathology (edited by A. E. Elliss).Academic
Press, LOndon,pp.

107‑117.

山形県において 分離された自発凝集性をいAe

1077‑1084.

salmonicida. Mar.

Rev., 40, 12‑17.
Ward, P. D., C. A. Waters, and K. J. Sweeney (1985):「

oomonas

示さな

salmon@ ， cida

野村 哲 ―・古木守 ・茂木省三・絵面 良田

山形県で分離された23株および北海道で分離された10株のA 。 Omon0$50lm。 血 Cid0について，液体培地での自発凝集性， CO0maSSiebrM 如tblu 。 を添加した寒大培地上に発育したコ p 二―への色素の吸着およびSDS‑PAGE
によるA‑layerのタソ" ク 組成を検討した．山形県で分離「した株中4株は液体培地中で弱い自発凝集性を示し寒大培地に出現したコ p 二―は主として色素の吸若を示さな
い コ p 二―であったが，―部のコ p 二―は吸着を示した．

他のM株 は全て自発凝集性を示さず，出現コ p 二―に色素の吸着は認められなかった．
SDS ， PAGE では山形県での 分離株戊 北海道での分離株に見られた50kDa ・の主要な タソ" ク 質の存在は認められないか，微量であった・

以上の結果から山形県での分離株@こは自発凝集性を示さないAA.salmonicidaが存在することが明らかになった．