ハタハタにみられた非定型Aeromonas salmonicida感染症

誌名誌名 魚病研究

ISSNISSN 0388788X

巻/号巻/号 452

掲載ページ掲載ページ p. 92-95

発行年月発行年月 2010年6月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

f.¥l1)耳石)f究 Fish Pathology， 45 (2)， 92-95， 2010. 6

Atypical Aeromonas salmonicida

Infection in Sailfin Sandfish

Arctoscopus japonicus

Shinpei Wada 1

へKazukiOzeki1， Osamu Kurata 1，

Kishio Hatai1， Aya Ishida

2 and Kenjiroh Ui

2

1 Laboratory of Fish Diseases， Nippon Veterinary and Life

Science University， Tokyo 180-8602， Japan

20ga Aquarium GAO， Akita 010-0673， Japan

(Received April19， 2010)

ABSTRACT -In 2009， a disease with cumulative mortality

of 52.5% was observed in a captive population of sailfin

sandfish Arctoscopus japonicus reared at an aquarium in

Japan. The affected fish had no apparent lesions on the

body surfaces， with the exception of skin abrasions on the

lower jaw冒 Thetrunk kidney was swollen and bore white

miliary nodules. The most prominent histopathological fea-

ture of the diseased fish was numerous bacterial colonies in

the internal organs. However， no inflammatory cellular

response was apparent. Single species of bacteria was

isolated from the trunk kidney and spleen， and identified as

atypical A. salmonicida based on the biochemical and sero-

logical characteristics

Key words: Arctoscopus japonicus， atypical Aeromonas

salmonicida， sailfin sandfish

Sailfin sandfish Arctoscopus japonicus are scale-

less， nocturnal marine cold-water fish that are found in

the northern part of Japan. A. japonicus is an impor-

tant commercial species for fisheries and constitutes a

part of the local cuisine of Akita Prefecture in Japan.

The Oga Aquarium GAO has been exhibiting the spe-

cies for several years as part of efforts to provide histori-

cal and regional education， especially to younger

residents. In 2009， a disease outbreak occurred in a

captive population of A. japonicus reared in a tank at

the Oga Aquarium GAO. The disease was character幽

ized by bacterial septicemic lesions in the internal

organs and aUributed to an atypical Aeromonas

salmonicida infection. Infections of non-salmonids

with atypical A. salmonicida have been reported in a

variety of marine and freshwater fish species in the

world1). In Japan， atypical A. salmonicida infection has

also been described in some non-salmonid marine spe-

cies including shotted halibut Eopsetta grigorjewi2)，

* Corresponding author E-mail: swada@nvlu.ac.jp

。2010The Japanese Society of Fish Pathology

greenling Hexagrammos otakii， Japanese flounder

Paralichthys olivaceus， Schlegel's black roclくfish

Sebastes schlegeIP.4)， and marbled sole Pleuronectes

yokohamae5). The present study describes the clinical

signs， gross features and histopathology of the dis-

eased sailfin sandfish， as well as identification of the iso胴

lated strains.

Materials and Methods

Fish rearing

This captive population of A. japonicus had origi-

nated from artificial seed produced by the Oga

Aquarium GAO with brood fish captured in the Japan

Sea in December 2008. In February 2009， 973 fish

had been transferred to the tank to be exhibited. The

fish were housed in a rearing tanlく(20m3) with an open

circulation system using filtrated natural seawater. The

rearing water was aerated and chilled to maintain the

temperature within the range of 4.20C to 5.20C. The fish

were fed krill (Order Euphausiacea) and sand eel

Ammodytes personatus.

Bacteriological examination

Bacteriological swabs were taken from the trunk kid-

ney and spleen of seven diseased fish， plated onto

heart infusion agar (Nissui) supplemented with 10%

FBS (JRH Bioscience) and， then incubated at 200C for

2 days. Single colonies were picked and subcultured.

The morphological and biochemical characteristics of

the isolates were tested by the standard methods6) and

using the API20E (bioMerieux).

Histopathological and immunological examinations

Histopathological examinations were performed on

five of the infected fish. The samples were fixed in

10% phosphate輔 bufferedformalin solution， decalcified in

ethylenediaminetetraacetic acid (EDTA) with 5% foト

malin， and then processed routinely for histology. The

sections were stained with hematoxylin and eosin

(H&E) and Gram stains.

Fluorescent-antibody technique was performed to

detect A. salmonicida antigens using some selected

samples. After deparaffinization， the sections were pre-

treated in 10 mM citrate-buffered solution (pH 6.0) for 10

min at 1100C for antigen retrieval. After washing with

phosphate.・bufferedsaline (PBS)， the sections were

incubated with 5% FBS in PBS for 30 min to reduce

nonspecific background. Immunofluorescence staining

was performed using a rabbit anti-A. salmonicida serum

raised against a typical strain (ATCC 14174) of A.

salmonicida as a primary antibody and FITC conjugated

anti-rabbit immunoglobulins goat serum (Invitrogen) as

a secondary antibody.

A. salmonicida infection in sailfin sandfish

Results

Clinical history and gross features

Individuals in this captive population of A. japonicus

were hypersensitive to external stimuli such as flash-

lights， sounds and vibrations， and to respond by darting

towards the tank walls. This resulted in moderate skin

abrasions， especially on the lower jaw and carapace，

which were associated with swelling and reddening

prior to mortality in some cases.

Clinical signs of disease were initially observed in

the population in mid-March 2009. Diseased fish that

were isolated from the tank mates， showed appetite

reduction， rapid breathing， poor swimming ability， and a

Fig. 1. Gross features of diseased A. japonicus. The lower jaw and carapace showed abrasions with reddening (arrows)

93

tendency to congregate near the surface of the water

and the overflow pipe， but no ulcerative lesions were

observed on the body surfaces (Fig. 1). The trunk kid-

ney of the diseased fish was swollen and bore white mi卜

iary nodules (Fig. 2). In some cases， the spleen also

showed swelling and large white nodules. A low-to-

moderate mortality rate (1-20 dead fish per day) was

recorded for 3 months starting in mid-March 2009. By

the end of May 2009， only 461 fish survived in the tank

(cumulative mortality; 52.5%). The individuals sam-

pled for the present study ranged from 10.4 cm to 18.6

cm in body length

Fig. 2. Trunk kidney of diseased A. japonicus. Swelling was apparent and the organ bore white miliary nodules (arrows)

Table 1. 8iochemical characteristics of present isolates and some A. salmonicida isolates

Present r.1 isolates

Atypical A. salmonicida from marine fish

EK-9202*2 isolates*3 SeOY -9401科 AAS0301*5 MH7663柑

A. salmonicida subsp

salmonicida*7

Gram stain Motility Form Catalase Cytochrome oxidase OFtest 8rown pigment Auto-aggregation Indole production Gelatinase Arginine dihydrolase Lysin decarboxylase s-Galactosidase Acid from

ND*8

ND Short rod Short rod Short rod Short rod Short rod

+ + + + +

+ + + + + F*9 F F F F *10 W/- W + W

+ + + + +

+ + + + +

+ + + + +

+ + + ND + + + +

+ + + +

L-arabinose + +

+

+ sucrose Reactivity on Anti-A. salmonicida rabbit serum + +

Short rod

++Fr

ト

DL沖

D

4NWN

+ +

+ + d*11

d

+ +

+ + +

+

+

+

+ + + + +

*1 AII isolates showed the same characteristics. *2 Nakatsugawa， 1994. 時 lidaet al.， 1997. 料 Izumikawaand Ueki， 1997. 時 Kumagaiet al.， 2006 柑 Treasureret al.， 2007. 羽 Holtet al.， 1994 柑 Notdescribed. *9 Fermentative. *10 Weakly positive. 斗1111-89% of strains are positive.

94 S. Wada， K. Ozeki， O. Kurata， K. Hatai， A. Ishida and K. Ui

8acterial characterization

Bacteria were isolated from all seven of the dis-

eased fish. The isolates showed similar characteristics.

They were non-motile， Gram-negative， rod-shaped，

oxidase-positive， catalase-positive and glucose-ferment-

ing， and shared some other biochemical properties.

They also reacted to anti-A. salmonicida rabbit serum in

the immuno・fluorescentstaining. In comparison with

the A. salmonicida subsp. salmonicida (typical A. sal-

monicida)6)， the present isolates showed differences in

indole production and acid production from sucrose

(Table 1). The isolates also differed from other atyp卜

cal A. salmonicida strains from marine fish in some bio-

chemical characteristics (Table 1).

Histopathological features

The most prominent histopathological feature of the

diseased fish was the presence of numerous， variable

sized bacterial colonies in the internal organs including

the heart， trunk kidney， spleen， liver (Fig. 3)， gills， intes-

tine and gonads. The bacterial colonies occupied a

large proportion of the internal organs. In the heart

and trunk kidney in particular， bacterial colonies with

relatively little surrounding necrosis largely replaced the

parenchyma and interstitium of the organs. Slight-to-

moderate necrosis of the host tissue was observed

around the colonies in the other internal organs; how司

ever， no inflammatory cellular response was apparent，

with the exception of the presence of a small amount of

Iymphocyte infiltration. No bacterial lesions were

observed in the central nervous system of the diseased

fish. The epidermis of the infected fish sloughed 0仔at

the lower jaws and carapace， causing degenerative

changes of the stratum spongiosum which were associ-

ated with numerous colonies of bacteria (Fig. 4a). The

bacteria were identified as Gram-negative， short

rod-shaped bacteria， and stained positively with anti-A.

salmonicida serum (Fig. 4b).

Discussion

The isolates from the diseased sailfin sandfish

were identified as atypical A. salmonicida based on

their bacteriological characterization tests. Their bio-

chemical characteristics differed from those of other

atypical A. salmonicida strains isolated from marine fish

Although the pathogenicity of the present atypical

A. salmonicida isolates to sailfin sandfish remains to be

examined， it is thought that the present mortality was

due to atypical A. salmonicida infection. Most atypical

A. salmonicida infections in non-salmonid marine fish

have been reported to manifest predominantly as skin

Fig. 3. Histopathological sections of A. japonicus infected with atypical A. salmonicida showing numerous， variably sized bacterial colonies in the internal organs. In the heart and trunk kidney in particular， the parenchyma and interstitium were almost replaced by large bacterial colonies， with little surrounding necrosis a. heart b. trunk kidney c. spleen d. liver H&E Bars = 100μm

A. salmonicida infection in sailfin sandfish 95

Fig.4. Histopathological sections of the epidermis at the lower jaws of the affected A. japonicus. The epider-mis was associated with a number of colonies of short rod-shaped bacteria (a) that were stained positively with anti-A. salmonicida serum (b). a. H&E Bar = 30 μm b. Immuno-staining with anti-A. salmoniocida serum Bar = 50μm

diseases (ulceration or erosion) 1-4). Their freshwater

counterparts， such as erythrodermatitis of common carp

Cyprinus carpi07)， ulcer disease of goldfish Carassius

auratus8)， and head ulcer disease of eels Angui/la ros-

9-14) trata， A. angui/la and A. japonica~'~I ， presented

primary skin ulceration associated with subsequent sep-

ticemia in some cases.

The present disease was characterized by prom卜

nent and severe septicemic lesions in the internal

organs， with no major host defence reaction or skin

ulcers. Similar histopathological features， character-

ized by septicemic lesions in the internal organs， were

previously reported in an outbreak of atypical

A. salmonicida infection in cultured P. yokohamae5).

Treasurer et a/. (2007)15) reported that cultured haddock

Melanogrammus aeglefinus infected with atypical

A. salmonicida showed septicemic pathology character-

ized by severe liquefaction necrosis and the formation

of bacterial colonies in the internal organs， and a signifi-

cant inflammatory response that mainly comprised

macrophage-like cells was also observed， whereas a

host defence reaction was not apparent in this study.

Bacterial myocarditis associated with large bacterial

colonies， with little surrounding necrosis， which was

almost pathognomonic for furunculosis， was reportedly

caused by typical A. salmonicida infection in some

salmonids16). The disease described here therefore

presented pathological features including a relatively

intact body surface without ulcerative lesions， severe

septicemic lesions corresponding to salmonid furunculo-

sis with little surrounding necrosis， and a weak inflam-

matory response. In the present population， the skin

abrasions associated with swelling and reddening of the

lower jaw might have acted as an “portal of entry"，

because the lesions became apparent prior to the mor-

tality， and they were positive for immuno-fluorescent

staining with anti-A. salmonicida serum. The clinical

history suggested that the diseased fish were exposed

to high levels of stress due to external stimuli. The fish

might therefore have been immuno-compromised and

suffered septicemia lacking sufficient defence

mechanisms

Acknowledgements

We wish to thank the Japan Fisheries Resource

Conservation Association for providing anti-A. salmonicida

rabbit serum.

References

1) Wiklund， T. and 1. Dalsgaad (1998): Dis. Aquat. Org.， 32， 49-69. 2) Nakatsugawa， T. (1994): Fish Pathol.， 29， 193-198 (in Japanese with English abstract). 3) lida， T.， C Sakata， H. Kawatsu and Y. Fukuda (1997): Fish Pathol.， 32， 65-66 (in Japanese with English abstract). 4) Izumikawa， K and N. Ueki (1997): Fish Pathol.， 32， 67-68 (in Japanes with English abstract). 5) Kumagai， A.， K. Sugimoto， D. Itou， T. Kamaishi， S. Miwa and T. lida (2006): Fish Pathol.， 41，7-12 (in Japanese with English abstract). 6) Holt， J. G.， N. R. Krieg， P. H. A. Sneath， J. T. Staley and S. T. Williams (1994): In “Bergey's Manual of Determinative Bacteriology Ninth Edition" (ed. by J. G. Holt， N. R. Krieg， P. H. A. Sneath， J.丁目 StaleyandS. T. Williams)， Williams and Wilkins， Baltimore， pp. 190-191， pp. 253-255. 7) Bootsma， R.， N. Fijian and J. Blommaert (1977): Vet. Arh.， 47， 291-302. 8) Saito， M.， T. Nakamura and K. Takahashi (1975): Fish Pathol.， 9， 179-186 (in Japanese with English abstract). 9) lida， T.， K. Nakakoshi and H. Wakabayashi (1984): Fish Pathol.， 19， 109-112 (in Japanese with English abstract). 10) Kitao， T.， T. Yoshida， T. Aoki and T. Fukudome (1984): Fish Pathol.， 19，113-117. 11) Kitao， T.， T. Yoshida， T. Aoki and T. Fukudome (1985): Fish

Pathol.， 20， 107-114. 12) Ohtsuka， H.， T. Nakai， K. Muroga and Y. Jo (1984): Fish Pathol.， 19，101-107 (in Japanese with English abstract). 13) Noga， E. J. and H. A. Burkhoff (1990): Fish Pathol.， 25， 127-132. 14) Dalsgaad， 1. (1994):丹skHav， Skr. Danm. Fiskeri. Havunders.， 15， 35-38. 15) Treasurer， J. w.， T. H. Birkbeck， L. A. Laidler and D. 1. Cox (2007): J. Fish

D低，30，313-318. 16) Ferguson， H. W. (2006): Cardiovascu-lar system， In “Systemic Pathology of Fish， 2nd edition" (ed. by H.W目 Ferguson)，Scotian Press.， UK.， pp. 141-16士

カンパチ，マダイおよびマウスに対する Mycobacterium

marinumの病原性

S. Weerakhun .和田新平・畑井喜可1if~佐野文子・仁部玄通・平江多績

筋肉内接種による人為感染試験の結果，ブリ病魚、由来

Mycobacterium marinum NJB 0419株のカンパチおよび

マダイに対する病原性が確認された。動物由来感染症の

観点から， BALB/cマウスに対する本産iの病原性を血管内

及び皮下接穣により検討した結来，病理組織学的変化や

組織中の菌の存在が認められず，再分離もされなかった。

本菌は陪乳類には病原性を示さないと考えられた。

魚、病研究， 45 (2)， 88-91 (2010)

ハタハタ!こみ5れた非定型 Aeromonassalmonicida

感染症

和田新王子・尾関一輝・倉田 修・畑井喜司雄

石田 綾・宇井賢二郎

2009年 3月から 5月に，秋田県下の水族館で飼育され

ていたハタハタに異常な死亡(累積死亡率52.5%) がみ

られた。病魚、にはスレによると考えられる下顎のびらん

を除いて外部症状は認められなかったが，剖検すると体

腎が腫大し粟粒結節がみられた。病理組織学的には，内

臓諸組織に著しい細菌の増殖像(集落)が観察された。

体腎と牌臓から単一の細菌が分離され，生化学的および

I削青学的性状から非定型A.salmonicidaに同定された。

f手jお研究， 45 (2)， 92-95 (2010)