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: [email protected]
。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
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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)