Acremonium sp.に対する各種抗真菌剤のin vitroおよびinvivoでの効果

誌名誌名 魚病研究

ISSNISSN 0388788X

巻/号巻/号 453

掲載ページ掲載ページ p. 109-114

発行年月発行年月 2010年9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

魚病研究円shPathology， 45 (3)， 109-114， 2010. 9 。2010The Japanese Society of Fish Pathology

In vitro and In vivo E官icacyof Antifungal Agents against Acremonium Sp.

Pham Minh Duc1ペShinpeiWada¥ Osamu Kurata

1 and Kishio Hatai

1*

1 Laboratory of 丹'shDiseases， Nippon Veterinary and Life Science Universit.μ Tokyo 180・8602，Japan

2College of Aquaculture and丹'sheries，Cantho University， Can Tho City， Viet Nam

(Received April 8， 2010)

ABSTRACT -Acremonium sp. NJM 0672， isolated from diseased mantis shrimp Oratosquilla oratoria， was susceptible in vitro to three kinds of antifungal agents: voriconazole， amphotericin B and terbinafine hydrochloride. Voriconazole was selected to treat kuruma prawn Penaeus japonicus， which had been intramuscularly injected with 0.1 mL of 5.0 x 104 conidiaJmL of Acremonium Sp. Voriconazole was admin-istered orally at doses of 6 and 2 mg/kg body weight per day for 7 consecutive days， or intramuscularly injected at doses of 4 and 2 mg/kg body weight per day for 3 consecutive days. Both treatments were begun at 6 h after injection of the conidial suspension. The gross features， mortality and histopathologi-cal findings demonstrated that voriconazole was an efficient antifungal agent against Acremonium sp.

Key words: Acremonium sp.， antifungal agent， voriconazole， Penaeus japonicus， kuruma prawn，

Oratosquilla oratoria

Studies on the susceptibility of fungi to antifungal

agents have been reported previously (McGinnis and

Pasarell， 1998; Espinel-Ingroff， 2001; Kamai et al.，

2002; Vitale et al.， 2003; Serena et al.， 2003; Makimura

et al.， 2004; Koga et al.， 2006). Some antifungal agents

such as voriconazole， itraconazole， amphotericin B and

flucytosine have been used to control fungal infection in

humans and animals (Espinel-Ingroff， 2001; Vitale et al.，

2003). In vit.ro susceptibility test of anamorphic fungi is

helpful for understanding the susceptibility of the organ-

ism to antifungal agents. The minimum inhibitory con-

centrations (MICs) of antifungal agents against some

fungi may be a useful guideline for selecting effective

drugs.

The anamorphic fungus Acremonium sp. is a seri-

ous pathogen that causes gill disease in mantis shrimp

Oratosquilla oratoria (Duc et al.， 2009). The severity of

this fungus has been confirmed by pathogenicity tests

on healthy mantis shrimp (Duc and Hatai， 2009). How-

ever， infections with the anamorphic fungus have not

been reported in kuruma prawn Penaeus japonicus，

which is cultured in the same area with infected wild

mantis shrimp. Antifungal agents for the treatment of

fungal infection in humans (Supparatpiyo and Schlamm，

2007; Hariprasad et al.， 2008) and animals (Rochette et

al.， 2003; Sponsel et al.， 2006) are well known， but stud-

* Corresponding author E-mail: khatai0111@nvlu.ac.jp

ies on the treatment of fungal infections using antifungal

agents are rare in aquatic animals (Cabanes et al.，

1997). Because of the potential risks of disease out-

breaks due to the fungal species in kuruma prawn

farms in the future， we principally attempted to evaluate

the effectiveness of some antifungal agents against

Acremonium sp.， as well as the clinical effectiveness of

voriconazole against experimental infection in kuruma

prawn in this study.

Materials and Methods

Antifungal agent

Eight antifungal agents were used in this study: flu-

conazole and amphotericin B (Wako Pure Chemical

Industries Co.， Ltd.)， 5・fluorocytosine(Tokyo Chemical

Industry Co.)， terbinafine hydrochloride (Novartis

Pharma)， micafungin (Astellas Pharma Co.， Ltd.)，

miconazole (Mochida Pharmaceutical)， itraconazole

(Janssen Pharmaceutical) and voriconazole (Pfizer

Pharma Co.， Ltd.). Stock solution of water-insoluble

agents (fluconazole， amphotericin B， 5・fluorocytosine，

terbinafine hydrochloride) was prepared in dimethyl sul-

foxide at a concentration of 1，600μg/mL， and that of

water-soluble agents (micafungin， miconazole， itracona-

zole， voriconazole) in sterile distilled water (D.W.) at a

concentration of 5，120μg/mL. The final concentra開

tions of the antifungal agents of 0.031 to 64μg/mL

were produced by serial two-fold dilutions in combina-

110 P. M. Duc， S. Wada， O. Kurata and K. Hatai

tion with ten-fold dilution.

Fungal strain and inoculum preparation

Acremonium sp. NJM 0672， isolated from the gills

of diseased mantis shrimp (Duc and Hatai， 2009) was

used in this study. Stock inoculums were prepared as

described in Approved Standard M38-A， NCCLS， Wayne，

PA， pp22 (16) (NCCLS M38・A)(National Committee for

Clinical Laboratory Standards， 2002)， which is approved

as a standard method for broth dilution antifungal sus-

ceptibility testing of filamentous fungi. Acremonium sp.

NJM 0672 was incubated at 250C for 10 days on PYGS

agar (Bian et al.， 1979). The conidial suspension was

made by adding 10 mL of sterile saline (0.85% NaCりon

the agar plate containing fungal colonies， and crushing

the surface of the colonies with a sterile loop to sus-

pend conidia. The conidia and mycelia suspension

was then filtered through two sterile layers of sanitary

medical gauze to obtain a conidial suspension. The

number of conidia was determined using a hemocytome-

ter and the concentration was adjusted to 5.0 X 105

conidia/mL for in vitro susceptibility tests， and 5.0 x 104

conidia!mL forわ vivoexperiment.

Medium

RPMI 1640 (10.2 9 powdered RPMI 1640 medium，

0.3 9 L-glutamine and 34.53 9 MOPS 3・N-morpholino

propanesulfonic acid in 1 L of D.W.) was used as a

basic medium for in vitro susceptibility tests. The

medium was adjusted to pH 7.0 using 1 N sodium

hydroxide， filtered with a 0.45・μmsterile membrane fi卜

ter (47 mm in diameter) (Toyo Roshi Co.)， and then

stored at 40C until use.

Minimum inhibitory concentration (MIC) and minimum

fungicidal concentration (MFC)

In in vitro susceptibility tests， the broth microdilution

method was conducted as described in NCCLS M38-A.

Each well of 96・wellcell culture plate (Corning) contain-

ing 100μL of the serially diluted drug with RPMI 1640

medium was mixed with 100μL of conidial suspension.

Thus， the final concentrations of antifungal agents in

these wells ranged from 0.016 to 32μg/mL. Two con-

trol wells were used-one containing 50μL of dimethyl

sulfoxide with 50μL of sterile artificial seawater (ASW)

with 35%0 (w/v) salinity (Marine Aquarium Salt， Nisso

Co.， Ltd.)， and the other containing 100μL of sterile

ASW. These two wells were then added with 100μL

of the conidial suspension. The control wells were pre-

pared to determine fungal growth of the strain in marine

environment， with or without dimethyl sulfoxide. Micro-

dilution trays were incubated at 250C， and examined at

72 h to determine MICs against Acremonium sp. NJM

0672 according to the method described in NCCLS

M38・A. MFCs， the minimum drug concentration that

resulted in either no growth or fewer than three colonies

(99.9% killing)， were determined using the method

reported in Espinel開 Ingro汗(2001).

Kuruma prawn

A total of 120 kuruma prawns with an average body

weight of 10.5 9 was obtained from a private farm in

Kagoshima Prefecture， Japan， in July 2008， and

stocked in six 20・Ltanks. The kuruma prawns were

reared in ASW with 35%0 (w/v) salinity， and acclimatized

for 1 week before experimental infection. They were

fed daily with 2% body weight of commercial food.

Water temperature was kept at 16-180C and pH 創立5

during the course of the experiment. Thirty healthy

kuruma prawns were selected for experimental infection

and then divided into six equal groups in six separate

tanks. The kuruma prawns were fed daily with 2%

body weight of commercial food before and during

experiment.

Treatment of kuruma prawns infected with Acremonium

sp. NJM 0672

The experimental infection was conducted at the

Laboratory of Fish Diseases， Nippon Veterinary and Life

Science University， from July to August 2008. Each

prawn was intramuscularly injected with 0.1 mL of the

conidial suspension of 5.0 x 104 conidia!mL. Voricona・

zole was selected and used in this experiment based on

in vitro susceptibility tests of antifungal agents. The

drug was diluted with sterile saline (0.85% NaCI) and

used for intramuscular injection and to make medicated

food. Commercial food used to maintain the prawns

was mixed with the diluted drug， then dried in room

temperature. Two groups of prawns were orally admin-

istered a high (6 mg/kg body weight!day) or low (2

mg/kg body weighVday) dose of voriconazole， for 7 con-

secutive days， starting 6 h after injection of the conidial

suspension. In other two groups， kuruma prawns were

intramuscularly injected with a high (4 mg/kg body

weighVday) or low (2 mg/kg body weight!day) dose of

voriconazole (final volume， 0.1 mL) for 3 consecutive

days， starting 6 h after the conidial injection. The dos-

age of drug and administration times were decided

based on the results of preliminary experiments. In a

negative control group， each kuruma prawn was only

injected with 0.1 mL of sterile saline (0.85% NaCI) and

was not treated with the drug. In non-treatment group，

each kuruma prawn was injected with the conidial sus-

pension， and was not treated with the drug. Clinical

signs including inactive swimming and black gill lesions

and mortality were recorded for 25 days. Moribund， as

well as freshly dead prawns were collected for histopa-

thological examinations and fungal re-isolationin during

the course of experiment. Fixed samples in 10%

phosphate buffered formalin solution were routinely

processed to paraffin sections and stained with methe-

namine silver nitrate圃 Grocott'svariation， counter-stained

Antifungal agents against Acremonium sp.

with hematoxylin and eosin (Grocott-H&E). Histopa-

thological characteristics were examined under a light

microscope. Fungal re-isoation with moribund and

freshly dead prawns was performed by inoculating small

pieces of gill filaments of the prawns on PYGS agar

plates. These agar plates were incubated at 250C for

10 days， and the isolates were identified with their mor-

phological characteristics by slide culture method (Duc

et al.， 2009). Fungal re-isolation was also attempted

with all of the prawns showing clinical signs and the

prawns in negative control group at the end of

experiment.

Results

In vitro effectiveness of antifungal agent

The antifungal activities of the eight agents are

shown in Table 1. Three antifungal agents (voriconazole，

amphotericin B and terbinafine hydrochloride) were

highly active against NJM 0672. Both the MIC and

Table 1. In vitro activity of antilungal agents against Acremonium sp. NJM 0672

Acremonium sp. NJM 0672

Antilungal agents MICa MFCa

(μg/mL) (μg/mL)

Fluconazole >8 NT

amphotericin B 0.13 0.13

5-lluorocytocine > 32 NT

terbinaline hydrochloride 0.5 0.5

micalungin >8 NT

voriconazole 0.5 1.0

miconazole >8 NT

itraconazole >8 NT

MIC: minimum inhibitory concentration. MFC: minimum lungicidal concentration a MIC and MFC were determined at 72 h. NT: not tested

1

1

1

MFC of amphotericin B against NJM 0672 were 0.13

μg/mL. The MICs of terbinafine hydrochloride and vori-

conazole against NJM 0672 were 0.5μg/mL， and the

MFCs were 0.5 and 1μg/mL， respectively. There

were no differences of fungal growth in ASW and in

dimethyl sulfoxide diluted with ASW.

Treatment of kuruma prawns infected with Acremonium

sp. NJM 0672

Kuruma prawns in the norトtreatmentgroup showed

gill lesions (Fig. 1A)， which is consistent with those of

mantis shrimp experimentally injected with Acremonium

sp. NJM 0672 (Duc and Hatai， 2009). Inoculated fun-

gus was re-isolated from the gills of all kuruma prawns

with clinical signs (Table 2). By day 25， in the group of

fed with the medicated diet beginning 6 h after injection

of the conidial suspension， a total of four out of five

prawns were dead in the low dose group， while only

one out of five was dead in the high dose group. On

the other hand， no prawn was dead in the group

injected with voriconazole at 6 h after injection of conid-

ial suspension， in both the low and high dose groups by

day 25. In the non-treatment group， all prawns died by

day 25， while the negative control group remained alive

throughout the experiment. In addition， kuruma

prawns in the non-treatment group first began dying at

day 13 and the last prawn died at day 19 after injection

of conidial suspension. However， kuruma prawns

orally administered with the high dose of voriconazole

first began dying at day 19 and no mortality was

recorded after that until day 25. In the low dose group，

prawns first began dying at day 11 and in total of four

prawns were died by day 25

Numerous black spots were observed in the gills of

all kuruma prawns川 thenon-treatment group (Fig. 1 A

and Table 2)， however; a small number of black spots

were found in the gills of kuruma prawns fed with the

medicated diet (Fig. 1 B). AII kuruma prawns in low

dose group showed gross lesions in the gills， while

Fig. 1. Kuruma prawns intramuscularly injected with 0.1 mL 01 5.0 x 104 conidia/mL 01 Acremonium Sp. NJM 0672. A. A kuruma prawn in non-treatment group showing black gills at day 15 after injection. B. Black spots (arrow) in gills 01 a kuruma prawn injected with 4 mg voriconazole/kg BW/day. The kuruma prawn survived at 25 days after inoculation. C. Black spots (arrow) in gills 01 a kuruma prawn orally administered 6 mg voriconazole/kg voriconazole BW/day. The kuruma prawn sur-vived at 25 days after inocuation.

112 P. M. Duc， S. Wada， O. Kurata and K. Hatai

three out of five prawns showed the lesions in high

dose groups (Table 2). On the other hand， numbers of

kuruma prawn showed gross lesions in the gills in the

experiment of treatment with voriconazole by intramus-

cular injection were three out of five in low dose， and

two out of five in high dose， respectively (Fig. 1 C and

Table 2).

Histopathologically， numerous hyphae were

Table 2. Effectiveness 01 voriconazole in treating kuruma prawns inlected with Acremonium sp. NJM 0672

Treatment groups Negative control group Non-treatment group

Items T1 T2 T3 T4 T5 T6

Number 01 dead prawn 4/5*' 1/5 0/5 0/5 0/5 5/5

Number 01 inlected prawn 5/5 3/5 3/5 2/5 0/5 5/5

Number 01 prawn 5/5 3/5 3/5 2/5 0/5 5/5

showing clinical sign

Dearee 01 clinical sian ホ2 ++ + + + +++

T1: oral administration 01 2 mg/kg T2: aral administration 01 6 mg/kg T3: intramuscular injection 01 2 mg/kg T 4: intra-

muscular injection 01 6 mg/kg T5: intramuscular injected only with sterile saline (0.85% NaCI) T6: intramuscular

injected with conidial suspension and no drug treatment

*' Number 01 prawns with pasitive sign/total number 01 prawns

日 一 noblack lesion on gill lilaments +: slight degree 01 black lesions on gill lilaments ++: moderate degree 01 black

lesions on gill lilaments with inactive swimming +++・Severedegree 01 black lesions 01 gill lilaments with moribundity

Fig. 2. Histopathological lindings 01 kuruma prawns intramuscularly injected with 0.1 mL 01 5.0 x 104 conidia/mL 01 Acremonium sp.

NJM 0672. Grocott-H&E Bars = 30μm A. Numerous hyphae (arrows) observed in the gill lilaments 01 a kuruma prawn in

non-treatment group (at day 15). B. Hyphae encapsulated (arrows) in muscle at the injection site 01 a kuruma prawn in non-

treatment group (at day 15). C. A lew hyphae (arrows) in the gill lilaments 01 a kuruma prawn orally administered with 6 mg

voriconazole/kg BW/day (at day 25). D. A lew hyphae encapsulated (arrow) in the muscle at the injection site 01 a kuruma

prawn injected with 4 mg voriconazole/旬 BW/day(at day 25)

Antifungal agents against Acremonium sp. 113

observed in the gill filaments (Fig. 2A) and these

hyphae were encapsulated in the cellular aggregations

in the non-treatment group at day 15 (Fig. 2B). In con-

trast to the kuruma prawns of the non-treatment group，

only a few encapsulated hyphae were observed at day

25 in the gill filaments and/or muscle at the injection site

in the surviving kuruma prawns fed with the medicated

diet of 2 and 6 mg/kg in body weight /day (Fig. 2C)， and

in the surviving prawns intramuscularly injected with the

drug of 2 and 4 mg/kg in body weightlday (Fig. 20).

Discussion

Vitale et al. (2003) found that MICs of amphotericin

B were 0.25 to 2 mg/mL against other anamorphic fungi

such as Exophiala spinifera. The results in this study

were also in agreement with the results of McGinnis

and Pasarell (1998) and Espinel-Ingroff (2001)， who

found that voriconazole， amphotericin B and itracona開

zole were extremely effective against filamentous Asco-

mycetes and dematiaceous fungi. In light of these find-

ings， it was possible to consider that amphoterincin B，

terbinafine hydrochloride and voriconazole would be

candidates for the effective antifungal agents against

anamorphic fungi exhibiting pathogenicity to aquatic

crustaceans， as well as terrestrial vertebrates. How-

ever， more detailed studies should be needed for safe

usage of amphotericin B and terbinafine hydrochloride

from the potential risks of toxicity of their solvent， such

as dimethyl sufoxide used in this study.

In this study， we did not select amphotericin B and

terbinafine hydrochloride desolved in dimethyl sulfoxide

for in vivo experiment considering the potential risks of

toxicity of the solvent against kuruma prawns， because

it was reported that the presence of dimethyl sufoxide

could increase the sensitivity of freshwater crustaceans

to hepatotoxic cyanobacterial extracts (Orobniewska et

al.， 2004). The results of the experiment of treatment

with voriconazole by oral or intramuscular administra-

tion demonstrated that voriconazole could effectively

inhibit fungal growth in the kuruma prawns artificially

infected with Acremonium sp. NJM 0672. Voricona-

zole is a triazole antifungal medication that is generally

used to treat serious and invasive fungal infections，

such as invasive candidiasis， invasive aspergillosis and

certain emerging fungal infections. These are gener-

ally seen in patients who are immunocompromised.

Although more detailed toxicological examinations，

including studies on persistence in the crustaceans， as

well as toxicity to human being， are required for confir-

mation， our data suggest that voriconazole， especially

oral administration of the drug， could be an effective

and practical antifungal agent to control anamorphic fun-

gal infection in kuruma prawn without showing apparent

toxicity against the crustacean， thus it would be a prom-

ising chemical applied to aquatic invertebrates that are

consumed as food for human. Infections with the ana-

morphic fungus have not been reported in kuruma

prawn， which is cultured in the same area with infected

wild mantis shrimp; however， it is important to seek

effective methods to prevent and control the disease

outbreak. Because of the potential risks of disease out-

breaks due to the fungal species in kuruma prawn

farms in the future， more detailed studies should be

needed to search more effective and safe antifungal

gents.

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ベトナム・ニャチャン湾の卵・仔魚に内部寄生する渦鞭

毛虫 Ichthyodiniumsp.感染の長期変動

A. M. 8hadrin . 0.8. Pavlov . M. V. Kholodova

ベトナム・ニャチャン湾の卵・仔魚、に内部寄生する

Ichthyodinium sp.の感染を季節ごとに長期に渡り調査し

た。寄生虫の同定は形態と188rRNA塩基配列によって

行った。塩基配列は沖縄の種苗生産スジアラから報告さ

れたものと一致した。 1993年に初めて検出された後，感

染率は2004年までは増加し続け，その後.2007年まで高

位で推移し， 2008-2009年で減少したものの， 2010年に

再び急増した。感染率は，異なる分類群の魚、種間で差異が

見られた他，近縁な魚、種間でも大きな違いが見られた。

魚病研究， 45 (3)， 103-108 (2010)

種苗を放流した河川での冷水病の発生状況調査

熊谷明・縄田暁・谷合祐一

放流前の検査で、冷水病菌~'性の人工アユ種苗を放流し

ているにもかかわらず，毎年本病が発生している河川の

感染源を検討した。 2006，2007年に放流前の検査で本菌

が分離されなかった種苗を冷水病菌フリー用水で、成熟期

まで継続飼育した結果，両年とも陽性であったことから，

放流魚が保菌していたと考えられる。これらの放流後，

天然魚、と放流魚、における本病の発生状況を調査した結果，

両年とも 6~8 月に冷水病が発生したが，発病源は06年が放流魚、， 07年が天然魚と放流魚であった。成熟期には

90%以上のアユから本菌が検出された。

魚病研究， 45 (3)， 115-120 (2010)

PCRによるスクーチ力症の原因繊毛虫 Miamiensis

avidusの同定および検出

丹下菜穂子・宋準柴・北村真一

形態観察による同定が難しいスクーチカ症原因繊毛虫

Miamiensis avidusを特異的に検出するため， 88U rRNA

遺伝子を標的とした PCR法を確立した。本 PCRの検出

限界は DNA量にして 125fgであり，他のスクーチカ繊

毛虫 3種の DNAを増幅しなかった。感染したヒラメ群を

PCR検査した結果，発症魚の90%，未発症魚の50%が

陽性となり，特に脳で、の検出率が高かった。以上より，

本 PCRはM.avidusの同定および魚体からの迅速検出に

有効であることが明らかとなった。

魚、病研究， 45 (3)， 130-132 (2010)

ベタにみ5れた腎芽腫

E. D. Lombardini . M. Law . B. 8. Lewis

米国でベタ Bettasplendensの成魚に腎芽腫がみられ

た(2症例)。いずれの症例も，腫蕩塊は腎臓組織の90%

以上を占めていた。病理組織学的にはこれらの腫蕩は，

芽球細胞，未熟な問葉系組織および糸球体様構造を形成

する上皮性細胞より構成されていた。また。最初の事例

では嚢胞性の腫蕩塊が体腔の 2/3をおおい，他の事例で

は白色の腫蕩塊が頭蓋から背ひ、れにかけて広がっていた。

Wilms' Tumor-1診断用抗体との反応性は不明瞭であっ

た。本論文はベタにおける腎芽腫の初報告である。

魚病研究， 45 (3)， 137-139 (2010)

Acremonium sp.に対する告種抗真菌剤の invitroお

よび的 vivoでの効果

P. M. Duc・和田新平・倉田 修

畑井喜司雄

擢病シャコより分離された Acremoniumsp.は in

vitroで抗真菌剤，ボリコナゾール，アンホテリシン Bお

よび塩酸テルビナフインに感受性を示した。 Acremonium

sp.に人為感染させたクルマエビにボリコナゾールを経口

および筋肉内投与した結果，肉眼所見，死亡率および病

理組織学的所見より，本薬剤は Acremoniumsp.に対し

て有効な抗真菌剤であることが示された。

魚病研究， 45 (3)， 109-114 (2010)

ヒラメ白血球包囲化反応における頼粒球およびケモ力イ

ンの活性

倉田修・ N.Kitancharoen・藤原篤志

中易千早・和田新平・畑井喜司雄

Ichthyophonus hoferiの多核球状体に対するヒラメ白

血球の的 vitro包囲化反応では，ベルオキシダーゼ陽性の

頼粒球が多数を占めていた。頼粒球は 1.hoferiに接着し

その周囲を取り囲んでいた。包囲化の過程において， 3種

類の CCケモカインおよび 1種類の CXCケモカインの

mRNA発現上昇が確認された。特に， CC-CLMおよび

|し8は頼粒球により産生され，異物包囲化の初期反応に

おける頼粒球の重要性が示唆された。

魚病研究， 45 (3)， 121-129 (2010)

シャコより分離された Plectos{Joriumoratosquillae

およびAcremoniumsp.のクルマエビlこ対する病原性

P. M. Duc・和田新平・倉田 修

畑井喜司雄

シャコより分離された不完全菌Plecわspo巾moraωsquillaeNJM 0662とAcremoniumsp. NJM 0672の分生子浮遊

液をクルマエビに筋肉注射した結果，いずれの菌もクル

マエビを死亡させた。病クルマエピの鯨には多数の黒色

点が出現し病理組織学的に概弁内および接種部周囲に

菌糸が観察され，それらは血球によって取り固まれてい

た。供試した菌はクルマエピに病原性を有することが示

された。

魚病研究， 45 (3)， 133-136 (2010)

168 rDI¥IAを標的とした Piscirickettsiasalmonis

検出用 PCRの改良

坂井貴光・熊谷 明・太田祐達

大迫典久・佐野元彦・飯田貴次

ピシリケッチア症の診断法として 168rDNAを標的と

する PCRによる原因菌の検出が報告されているが，その

プライマー領域の塩基配列に変異を生じている菌株が存

在する。そこで，新たに特異的なプライマーを設計した

結果，特異性と検出感度が向上した。さらに，本 PCRに

より人為感染魚の肝臓と腎臓から P.salmonisが検出され

た。従って，本 PCRはピシリケッチア症の診断に利用で

きると考えられた。

魚病研究， 45 (3)， 140-142 (2010)