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Phage Therapy against β-hemolytic Streptococcicosis of Japanese Flounder Paralichthys olivaceus

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We examined the therapeutic effect of Streptococcus iniae phages isolated from fish culture environments against experimental streptococcicosis of Japanese flounder Paralichthys olivaceus. Phage sensitivity tests with a double agar method revealed that 31 of 35 S. iniae strains from the flounder have a similar sensitivity to six phage isolates. In phage therapy experiments, fish were injected intraperitoneally (IP) with S. iniae PSi402 and 1 h later IP-injected with a mixture of two or four phage isolates, and observed at 25 degrees C for 2 wk. Mortalities of fish receiving phages were significantly lower than those of control fish without phage-treatment in all four trials. The effect of phage treatment was also demonstrated even at 24 h post-infection, when cell numbers of S. iniae were 10(7.4) and 10(4.5) CFU/g in the kidneys and brains of fish, respectively. However, as phage-resistant S. iniae were frequently isolated from dead fish in the phage-treated group, further investigations are required to establish phage therapy of the disease.
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Streptococcus iniae を原因とする
b
溶血性レンサ球菌
症は,日本をはじめ世界各地で種々の海産魚および淡水
魚で発生が報告されている(Kusuda and Salati, 1999
日本ではニジマス Oncorhynchus mykiss やアユ Pleco-
glossus altivelis などの淡水魚(Kitao et al., 1981
西・城, 1981およびヒラメ Paralichthys olivaceus
ブリ Seriola quinqueradiata などの海産魚(中津川
魚病研究 Fish Pathology, 42 (4), 181–189, 2007. 12 © 2007 The Japanese Society of Fish Pathology
ヒラメの
b
溶血性レンサ球菌症に対するファージ治療試験
松岡 学
1
*
・橋爪貴也
2
・神崎博幸
3
・岩本恵美
2
Se Chang Park
4
・吉田照豊
5
・中井敏博
2
2007 3 26日受付)
Phage Therapy against
b
-hemolytic Streptococcicosis of
Japanese Flounder Paralichthys olivaceus
Satoru Matsuoka
1
*
, Takaya Hashizume
2
, Hiroyuki Kanzaki
3
, Emi Iwamoto
2
,
Se Chang Park
4
, Terutoyo Yoshida
5
and Toshihiro Nakai
2
1
Ehime Prefectural Chuyo Fisheries Experimental Station, Iyo, Ehime 799-3125, Japan
2
Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima
739-8528, Japan
3
Saga Prefectural Genkai Fisheries Research and Development Center, Karatsu,
Saga 847-0122, Japan
4
College of Veterinary Medicine, Seoul National University,
Seoul 151-742, Korea
5
Faculty of Agriculture, University of Miyazaki, Miyazaki,
889-2192, Japan
(Received March 26, 2007)
ABSTRACT—We examined the therapeutic effect of Streptococcus iniae phages isolated from fish
culture environments against experimental streptococcicosis of Japanese flounder Paralichthys
olivaceus.Phage sensitivity tests with a double agar method revealed that 31 of 35 S. iniae
strains from the flounder have a similar sensitivity to six phage isolates.In phage therapy experi-
ments, fish were injected intraperitoneally (IP) with S. iniae PSi402 and 1 h later IP-injected with a
mixture of two or four phage isolates, and observed at 25°C for 2 wk.Mortalities of fish receiving
phages were significantly lower than those of control fish without phage-treatment in all four trials.
The effect of phage treatment was also demonstrated even at 24 h post-infection, when cell num-
bers of S. iniae were 10
7.4
and 10
4.5
CFU/g in the kidneys and brains of fish, respectively.How-
ever, as phage-resistant S. iniae were frequently isolated from dead fish in the phage-treated group,
further investigations are required to establish phage therapy of the disease.
Key words: bacteriophages, phage therapy,
b
-hemolytic streptococcicosis, Streptococcus iniae,
Paralichthys olivaceus, Japanese flounder
1
愛媛県中予水産試験場
2
広島大学大学院生物圏科学研究科
3
佐賀県玄海水産振興センター
4
ソウル大学獣医学部
5
宮崎大学農学部
*
Corresponding author
E-mail: matsuoka-satoru1@pref.ehime.jp
182 松岡 学・橋爪貴也・神崎博幸・岩本恵美・S. C. Park・吉田照豊・中井敏博
1983;酒井ら,1986;佐古, 1993で発生している。
特に養殖ヒラメにおいては本症はエドワジエラ症
Edwardsiella tarda 感染症)とともに重要疾病とされて
おり,問題点は化学療法の困難さにある。すなわち,罹
病したヒラメは摂餌活動が著しく低下するために薬剤の
経口投与効果が低い場合が多く,また本症の治療のため
に使用できる化学療法剤が 成分(塩酸オキシテトラサ2
イクリン,アルキルトリメチルアンモニウムカルシウム
オキシテトラサイクリン)に限られているうえに薬剤耐
性菌が出現するためである(松岡・和田,1996。一
このような医薬品の使用は養殖業者にとって経済的負担
が大きいうえに,食品の安全性の面からはできるだけ医
薬品を使用しない養殖方式が求められている。これらの
ことから,今後の持続的な養殖生産のためには,従来の
方法とは異なる視点から本症に対する対策を講じる必要
がある。
 近年,細菌感染症の治療に,細菌ウイルスであるバク
テリオファージ(以下ファージと略する)を用いる治療
法(ファージ療法)が注目され,獣医学や医学の分野で
その基礎研究が活発に行われ始めているBiswas et al.,
2002; Matsuzaki et al., 2003; Kutter and Sulakvelidze,
2004我々は水産増養殖分野におけるファージ療法に
ついて検討を進めており,これまでにブリのラクトコッ
カス症(Lactococcus garvieae 感染症)やアユの細菌性
出血性腹水病Pseudomonas plecoglossicida 感染症)
に対するファージ療法の有効性を報告してきたNakai
et al., 1999; Park et al., 2000; Park and Nakai, 2003
また最近になって海外からもカワマス Salvelinus
fontinalis のせっそう病に対するファージの有効性に関す
る報告がなされ(Imbeault et al., 2006魚類の細菌感染
症に対するファージ療法への関心が世界的に高まりつつ
ある。
 本研究では,ヒラメ養殖場の環境中から S. iniae に対
する溶菌ファージを分離し,それらのファージに対する
ヒラメ病魚由来 S. iniae 菌の感受性を調べるとともに
S. iniae を人為感染させたヒラメに対するファージの治療
効果について検討した。
材料および方法
海水およびヒラメからの S. iniae の分離
 ファージの分離と平行して,ファージの出現との関連
をみることを目的として,愛媛県大洲市2003年 月か5
2006年 月)および今治市2003年 月から200535
 月)のヒラメ養殖場で,養殖環境水および飼育中のヒ3
ラメ 才魚から S. iniae の分離を試みた。0
環境水として,陸上コンクリート水槽の排水(大洲市
または海面小割生簀周辺の海水(今治市)を毎月 回,2
 回あたり約 1 L 採取し,常温で愛媛県中予水産試験場1
の研究室に持ち帰った。このうち 250 mL をフィルター
ユニット0.45
m
mNalge Nunc Int.を使用して吸引
ろ過し,そのメンブレンフィルタ 10 mL の滅菌海水
の入った試験管に入れて約20秒間激しく攪拌した。この
懸濁液を試験水とし,AEアザイド・エスクリン)寒天
培地に塗抹して25°C で 日間培養した。出現したコロ7
ニーのうち,日本水産資源保護協会配布の抗 S. iniae
NIRA-2 株)家兎血清に対してスライド凝集試験陽性の
ものを S. iniae として計数した。
 ヒラメは,各養殖場で追跡調査する魚群を定めて毎月
 回外見上異常のみられない生魚10尾を採取し,氷蔵し1
て研究室に持ち帰った。それらの腎臓からトリプトソー
ヤ寒天培地(TSA:日水)を用いた画線塗抹法により細
菌の分離を行ない,25°C 48時間培養後に出現したコロ
ニーについて,上述の方法で S. iniae の同定を行なった。
分離された株の一部は,愛媛県および香川県の養殖ヒラ
メ病魚由来株とともに,ファージ分離のための宿主菌,
プラーク検出のための標示菌,およびファージ感受性試
験株として使用した(Ta ble 1
 なお,本研究で分離したものを含め供試した S. iniae
株について,Mata et al.2004の方法による乳酸酸化
酵素を標的とした PCR 試験を実施したところ,いずれの
株でも目的とするサイズの増幅産物870 bpが得られ
た。
ファージの分離
 既報の方法(松岡・中井,2004を用いて,集殖法に
より環境水から S. iniae ファージの分離を試みた。すな
わち,前述のフィルターユニット0.45
m
mでろ過し
た海水 100 mL を 倍濃度に調整した滅菌トリプトソー2
ヤブイヨンTSB:日水)100 mL に添加し,これに
2002年以前に分離された10株の S. iniae を宿主菌として
加えて25°C 24時間培養した。3,500 rpm 10分間遠心
分離した上清を 0.45
m
m でろ過しそのろ液における
ファージの有無を10株(2003年)または 株20045
よび2005年) S. iniae を標示菌Table 1として二重
寒天法(日高,1986;坂田・古川,2000により調べ
た。
ファージの形態観察
 単一プラークからの単離・培養を繰り返してクローン
化したファージ株について,透過型電子顕微鏡(日立
H-600A)によりファージ粒子の観察を行なった。
魚病細菌のファージ感受性試験
S. iniae39株)に加えて,比較のためレンサ球菌 L.
garvieae( 株) S. parauberis10株),またの他6
の魚病細菌として Vibrio anguillarum( 株)V. o r dalii2
183ヒラメレンサ球菌症のファージ療法
Table 1.Bacterial strains used in this study and their sensitivities against Streptococcus iniae phages
Sensitivity to phages Isolation
Strain
PSiJ 52PSiJ 51PSiJ 42PSiJ 41PSiJ 32PSiJ 31
YearLocationSource
Streptococcus iniae
++++++2001Ehime, JapanJapanese flounderIS-19
++++++2001Ehime, JapanJapanese flounderIS-21
a
++++++2001Ehime, JapanJapanese flounderIS-22
ab
++++++2002Kagawa, JapanJapanese flounderKRS-02-035
++++++2002Kagawa, JapanJapanese flounderKRS-02-036
ab
++++++2002Kagawa, JapanJapanese flounderKRS-02-042
a
++++++2002Kagawa, JapanJapanese flounderKRS-02-092
ab
++++++2002Kagawa, JapanJapanese flounderKRS-02-108
++++++2002Kagawa, JapanJapanese flounderKRS-02-111
a
++++2002Kagawa, JapanJapanese flounderKRS-02-118
++++++2003Ehime, JapanJapanese flounderPSi-301
a
++++++2003Ehime, JapanJapanese flounderPSi-302
a
++++++2003Ehime, JapanJapanese flounderPSi-303
ab
++++++2003Ehime, JapanJapanese flounderPSi-304
++++++2003Ehime, JapanJapanese flounderPSi-305
a
++++++2003Ehime, JapanJapanese flounderPSi-306
++++++2004Ehime, JapanJapanese flounderPSi-402
++++++2004Ehime, JapanJapanese flounderPSi-403
b
++++++2004Ehime, JapanJapanese flounderSIE-06
++++++2004Ehime, JapanJapanese flounderSIE-07
++++++2004Ehime, JapanJapanese flounderSIE-08
++++++2004Ehime, JapanJapanese flounderSIE-09
++++++2004Ehime, JapanJapanese flounderSIE-10
++++++2005Cheju, KoreaJapanese flounderSNUSI-05J2
++++++2005Cheju, KoreaJapanese flounderSNUSI-05J3
++++++2005Cheju, KoreaJapanese flounderSNUSI-05J4
++++++2005Cheju, KoreaJapanese flounderSNUSI-05J5
++++++2005Cheju, KoreaJapanese flounderSNUSI-05J7
++++++2005Cheju, KoreaJapanese flounderSNUSI-05J8
++++++2005Cheju, KoreaJapanese flounderSNUSI-05J9
++++++2005Cheju, KoreaJapanese flounderSNUSI-05J10
++++++2005Cheju, KoreaJapanese flounderSNUSI-05I15
2005Cheju, KoreaJapanese flounderSNUSI-05I16
2005Cheju, KoreaJapanese flounderSNUSI-05I17
2005Cheju, KoreaJapanese flounderSNUSI-05I18
++++++1985Mie, JapanYe llowtailYT-8504
++++++1992Miyazaki, JapanRainbow trout
b
-23
AT CC 29177
AT CC 29178
Lactococcus garvieae
AT CC 43921
AT CC 49156
2004Ehime, JapanJapanese flounderPSi 401
1995Ehime, JapanYe llowtailEU 206
1996Kagoshima, JapanYe llowtailKS 9601
1993Mie, JapanStriped jackMI 93005
Streptococcus parauberis
2005Ehime, JapanJapanese flounderPSP 501
2005Ehime, JapanJapanese flounderPSP 502
2005Ehime, JapanJapanese flounderPSP 503
2005Ehime, JapanJapanese flounderPSP 504
2005Ehime, JapanJapanese flounderPSP 505
2005Ehime, JapanJapanese flounderPSP 506
2005Ehime, JapanJapanese flounderPSP 507
2005Ehime, JapanJapanese flounderPSP 508
2005Ehime, JapanJapanese flounderPSP 509
2005Ehime, JapanJapanese flounderPSP 510
a,b
: used as indicator cells to isolate phages after enrichment culture (a: 2003, b: 2004 and 2005)
184 松岡 学・橋爪貴也・神崎博幸・岩本恵美・S. C. Park・吉田照豊・中井敏博
( 株)E. tarda( 株)Aeromonas hydrophila( 12 1
株)A. salmonicida( 株)Pseudomonas fluores-1
cence( 株)について,分離 S. iniae ファージ16
PSiJ31PSiJ32PSiJ41PSiJ42PSiJ51PSiJ52
に対する感受性をスポット法(坂田・古川,2000)に
り調べた。すなわち,TSB 25°C24時間培養した10
7
CFU/mL 濃度の菌液 0.4 mL 3 mL 1/3濃度の TSA
50°C で保温)と混合後,TSA 平板上に重層した。この
二重寒天平板上に 10
5
PFU/mL のファージ液を 滴(約 1
10
m
L)滴下し,25°C 24時間培養してプラークの有無
を観察した。ファージ液滴下部分に菌が発育しなかった
場合をファージ感受性と判定した。なお,供試した S.
parauberis 株については,長崎大学より分与された抗 S.
parauberisNUF934株)家兎血清に対するスライド凝集
試験により同定した。
ファージの in vitro 増殖抑制効果試験
S. iniae ファージ株PSiJ31PSiJ32PSiJ41
PSiJ42)について,試験管内での S. iniae 増殖抑制効果
を調べた10 mL TSB に,ブレインハトインフュー
ジョン寒天培地BHIA:日水)で25°C24時間培養し
S. iniae PSi402 株を終濃度 10
5
CFU/mL となるように
添加した。そこに MOImultiplicity of infection)が 10
0
10
–2
10
–4
となるように上述のファージ 株の単独または4
 株等量混合ファージ液をそれぞれ 10
m
L 添加した。対4
照区では,ファージ液の代わりに PBS 10
m
L 添加し
た。25°C で振とう培養し, ,61218および24時間1
後の吸光度(波長 600 nm)を測定した。
ファージによる治療試験
愛媛県中予水産試験場の隔離飼育施設においてS.
iniae を人為感染させたヒラメに対するファージ治療試験
回行なった。各試験には,魚体通過 回)させ51
S. iniae PSi402株の再分離菌を用いた。ファージ液
は,各ファージを二重寒天培地で S. iniae IS22株を宿主
菌として培養し,0.45
m
m のフィルターでろ過した後,
10
8
PFU/mL に調整した。試験 および では PSiJ31 12
PSiJ32 の  株のファージ混合液を,試験では235
PSiJ31PSiJ32PSiJ41 および PSiJ42 の 株のファー4
ジ混合液を使用した。
 試験 ∼ のうち試験 および では,ファージ接種14 1 2
区および対照区それぞれ 水槽100 L ポリアクリル1
円形水槽)に,試験 および ではそれぞれ 水槽に,34 2
平均体重 7.041.9 g のヒラメを2030尾ずつ収容した
TSA 25°C24時間培養した PSi402 株を,供試魚の腹
腔内に 0.1 mL/ 尾(10
5.4
10
7.7
CFU/ 尾)注射し,その 1
時間後にファージ液 0.1 mL を腹腔内注射した10
8.0
10
8.4
PFU/ 尾)対照区では供試魚に滅菌 TSB を同様に注
射した。その後,通気しながら流水で15日間飼育し,1
日に 回観察して死亡魚を取り上げるとともに,死亡魚2
および試験終了時の生残魚の腎臓から BHIA を用いて細
菌の分離を試みた飼育期間中の平均水温は24.2
25.0°C であった。
 試験 では,平均体重 56.4 g のヒラメに,PSi402 5
0.1 mL/ 10
5.4
CFU/0.1 mL腹腔内注射し,その12
時間または24時間後にファージ液を腹腔内に注射した
10
8.7
PFU/0.1 mL/ 尾)対照区では供試魚に滅菌 TSB
同量腹腔内注射して15日間観察した。各区 水槽を用い,2
 水槽あたり20尾を供試した。飼育条件は試験 ∼ と114
同様で,飼育期間中の平均水温は25.2°C であった。
 上記試験 と平行して,ファージ接種時における感染5
状態を知るため,菌接種後のヒラメ体内における S. iniae
の消長をみた。ヒラメ(平均体重 54.1 g)に S. iniae
PSi402 株を腹腔内注射し(10
5.4
CFU/ 尾) 12241
および48時間後の生魚と,菌液注射後 日目から 日目23
の死亡魚各 尾についてそれらの脳および腎臓を無菌的5
に摘出した。滅菌 PBS を加えて磨砕し適宜希釈して
BHIA  枚ずつに塗抹した25°C 48時間培養しS. 2
iniae コロニーを計数した。
統計処理
 試験結果の統計学的処理には
c
2
検定を用いp
0.05 を有意と判定した。
結     果
S. iniae の分離
 調査した養殖場においては,2003年の ∼12月に大洲9
市で S. iniae によるレンサ球菌症が発生し,その時の累
積死亡率は約15%であった。この期間中の外見上異常が
認められない魚(開腹すると,腎臓または脾臓が肥大し
ている個体もみられた)の腎臓における S. iniae 保菌率
は,60%( 月)20%(10月)40%(11月),および9
20%(12月)あった。これ以外に定期調査サンプルか
S. iniae が分離されたのは,大洲市における200411
(保菌率20%)2005年 月(10%)および12月(10 1
%)のみであった。一方,海水からは大洲市で2004年 7
月および11月に S. iniae が検出されたが100200
CFU/250 mL本症流行時には本菌は検出されなかっ
た。
ファージの分離
大洲市の養殖場の海水から200310月と11月,2004
12月および200510月に,また今治市の養殖場の海水
から200412月に S. iniae の溶菌ファージが分離され
た。このうち,代表的なプラークをクローニングして得
た 株のファージPSiJ31PSiJ32PSiJ41PSiJ426
185ヒラメレンサ球菌症のファージ療法
PSiJ51PSiJ52)を以下の実験に使用した(Table 2
分離ファージの形態
 分離されたファージの形成するプラークはいずれも円
形で,ほとんどのものが直径 ∼  mm であった(Fig. 12
1。また,電子顕微鏡で観察したファージ粒子は,いず
れも約 60 nm の正二十面体の頭部と, 160180 nm
のしなやかな尾部を有していた。これらの形態的特徴と
核酸として DNA を有することから,分離ファージは
Siphoviridae 科(Hendrix and Casjens, 2005に分類さ
れた。
魚病細菌のファージ感受性
分離したファージ 株に対する S. iniaeS. parauberis6
および L. garvieae の感受性を Table 1 に示したS. iniae
では,供試したヒラメ由来の35株中31株は,いずれの
ファージに対しても感受性を示した。残るヒラメ由来 S.
iniae 株のうち,香川県由来の 株はファージ 株中 162
に対して韓国済州島由来12株中の株はすべての3
ファージ株に対して感受性がなかった。また,他魚種由
来の S. iniae 株として供試したブリおよびニジマス由来の
S. iniae (いずれも 株)はすべてのファージ株に対して1
感受性を示したが,S. iniae type strainAT CC29177
および AT CC29178いずれもイルカ由来)は,いずれ
のファジに対しても感受性が認められなかった。一方,
S. parauberis および L. garvieae を含め,供試した S.
iniae 以外の魚病細菌 種24株は,いずれのファージにも8
感受性を示さなかった。
ファージの in vitro 増殖抑制効果
 ファージを添加していない対照区の濁度はS. iniae
増殖により12時間後には OD = 0.7 に達したFig. 2
ファージ添加区では,MOI = 1 から MOI = 10
–4
のいずれ
のファージ株単独添加区およびファージ 株の混合添加4
区とも12時間後まで濁度の上昇はほとんどみられず,
著な S. iniae 増殖抑制効果が認められた。しかし,18
間以降はどのファージ添加区でも菌の増殖が認められた。
Table 2.Source of Streptococcus iniae phages used in this
study
Isolation dateLocationStrain
2003. 10. 6Ozu, Ehime PrefecturePSiJ 31
2003. 11. 4Ozu, Ehime PrefecturePSiJ 32
2004. 12. 8Ozu, Ehime PrefecturePSiJ 41
2004. 12. 20Imabari, Ehime PrefecturePSiJ 42
2005. 10. 6Ozu, Ehime PrefecturePSiJ 51
2005. 10. 6Ozu, Ehime PrefecturePSiJ 52
Fig. 1.Plaques of S. iniae phage PSiJ41 in a double agar
plate.
Fig. 2.In vitro growth inhibition of Streptococcus iniae by
phages.S. iniae strain PSi402 and S. iniae phages
were inoculated at MOI = 1, 10
–2
, or 10
–4
, and shake-
cultured at 25°C for 24 h.The bacterial growth was
monitored optically.(Initial bacterial concentration:
10
5
CFU/mL) : individual phage strain (PSiJ31,
PSiJ32, PSiJ41, PSiJ42 from left); : mixed four
phage strains; : control (no phage)
186 松岡 学・橋爪貴也・神崎博幸・岩本恵美・S. C. Park・吉田照豊・中井敏博
培養24時間後にファージ添加区から菌を分離し,それら
のファージ感受性を調べた結果,いずれもそれぞれの
ファージに対して感受性を示さなかった。
ファージの感染防御効果
 計 回(試験 )の感染防御効果試験の結果を515
Ta ble 3Fig. 3 および Fig. 4 に示す。なお,試験 ∼ 35
における各区の死亡尾数の推移は 水槽でほとんど同じ2
であったため,生残率の推移を示した Fig. 3 および Fig.
4 ではそれら 水槽の平均値としてプロットした。2
 試験 ∼ では,全ての対照区で菌攻撃後 ∼ 日目14 12
から死亡が認められ, ∼ 日目に生残率は %となっ59 0
た。一方,菌攻撃後 時間目にファージを注射した試験1
区では,最初の死亡魚がみられたのは攻撃後 ∼ 日目24
とやや遅れ,1113日目以降には死亡魚はみられなかっ
た。15日間の観察期間中のファージ区の生残率は,試験
 が28.0%,試験 が33.3%,試験 が48.0%( 水槽1232
の平均),試験 が80.0%( 水槽の平均)で,いずれの42
試験においても対照区に比べて有意に高かった(Ta ble
3, Fig. 3試験終了時におけるファージ区生残魚の S.
iniae 保菌率(腎臓)は,試験 ∼ でそれぞれ42.9%,14
40.0%,41.7%および6.3%であった(Ta ble 3。死亡魚
から分離された菌についてファージ感受性を調べたとこ
ろ,対照区の分離菌はいずれのファージにも感受性が認
められたが,ファージ区の分離菌ではすべて感受性が認
められなかった。また,ファージ区生残魚からの分離菌
も,そのほとんどがファージ非感受性であった。
 試験 の生残率の推移を Fig. 4 に示した。対照区では,5
攻撃後 日目から死亡がみられ, 日後には85.0%が死23
亡し,15日後の生残率は5.0%となった一方,ファー
ジ接種区では,いずれも攻撃 日目から緩やかな死亡が3
続きそれらの生残率は12時間後ファージ接種区が
45.0%,24時間後ファージ接種区が32.5%と,いずれも
対照区に比べて有意に高かった。生残魚の保菌率は,12
時間後ファージ接種区が27.8%,24時間後ファージ接種
区が30.8%であった死亡魚および生残魚由来菌の
ファージ感受性は上述の試験 ∼ のそれと同じ傾向が14
認められた(Ta ble 3
 試験 と平行して行なったヒラメの腎臓および脳にお5
ける S. iniae 菌数の測定結果を Fig. 5 に示す。腎臓の菌
数は 時間後に10
3.6
CFU/ 尾であったが,その後急激に1
増加して12時間後に10
5.8
CFU/ 尾,24時間後に10
7.4
CFU/ 尾,さらに48時間後には10
8.1
CFU/ 尾にまで達し
た。方,脳では12時間後でも10
2.0
CFU/ 尾と少なかっ
たが,24時間後に10
4.5
CFU/ 尾となり,48時間後には腎
臓の菌数とほぼ同数の10
7.7
CFU/ 尾が検出された。死亡直
後の魚体では,さらにオーダー程度増加し,腎臓が1
10
9.3
CFU/ 尾,脳が10
9.6
CFU/ 尾であった。
Table 3.Phage treatment of Japanese flounder infected with Streptococcus iniae
Reisolation % of
S. iniae from survi-
vors (no. of fish
positive/examined)
Survival rate (%)
of fish (no. of fish
alive/examined)
Injection dose
(PFU/fish) of
phages (time after
S. iniae injection)
Injection dose of
S. iniae (CFU/fish)
Average body
weight (g)
Average water
temperature
(°C)
No. of
experiment
43 (3/7)28 (7/25)
*
10
8.2
(1 h)10
7.7
16.524.21
0 (0/25)0
40 (4/10)33 (10/30)
*
10
8.2
(1 h)10
6.3
7.024.32
0 (0/30)0
33 (4/12)48 (12/25)
*
10
8.0
(1 h)10
6.4
14.124.93
50 (6/12)48 (12/25)
*
10
8.0
(1 h)
0 (0/25)0
0 (0/25)0
0 (0/14)70 (14/20)
*
10
8.4
(1 h)10
5.4
41.925.04
11 (2/18)90 (18/20)
*
10
8.4
(1 h)
0 (0/20)0
0 (0/20)0
30 (3/10)50 (10/20)
*
10
8.7
(12 h)10
5.4
56.425.25
25 (2/8)40 (8/20)
*
10
8.7
(12 h)
29 (2/7)35 (7/20)
*
10
8.7
(24 h)
33 (2/6)30 (6/20)
*
10
8.7
(24 h)
ND5 (1/20)0
ND5 (1/20)0
*
, p < 0.05; ND, not done
187ヒラメレンサ球菌症のファージ療法
考     察
我々は前報で,愛媛県下のヒラメ養殖場における E.
tarda およびそのファージの周年変動を調べ,エドワジエ
ラ症の発生の有無にかかわらず高水温期を中心にヒラメ
E. tarda 保菌率は高く,また E. tarda ファージが養殖
環境水から高率に検出されることを示した(松岡・中井
2004同じく愛媛県下のヒラメ養殖場で行った今回の
調査では,レンサ球菌症流行時には S. iniae 保菌率は高
かったものの環境水中からの S. iniae S. iniae
ファージの検出率はともに低かった。我々がこれまでに
行なったアユ養殖環境からの病原細菌に対するファージ
の検出においても,P. plecoglossicida ファージは高頻度
に分離されたのにPark et al., 2000Flavobacterium
psychrophilum ファージの分離率は低かった(未発表)
このような養殖環境からのファージ分離率の違いは,自
然界における細菌の天敵としてのファージの存在意義を
考えると,興味深いところである。富栄養化し細菌数が
増加している沿岸域では,そこに存在するファージ数は
10
10
/L にも達するFuhrman, 1999ファージが細菌数
の調整者として重要な役割を担っているとすれば
Bergh et al., 1989; Proctor and Fuhrman, 1990,病気
Fig. 3.Phage treatment of Japanese flounder infected with Streptococcus iniae (Experiments 1–4).Fish were intraperitoneally (IP)
injected with phage mixture (Expt. 1, 2: PSiJ31, PSiJ32; Expt. 3, 4: PSiJ31, PSiJ32, PSiJ41, PSiJ42) 1 h after IP-injection
with S. iniae strain PSi402 (See Table 3). ○ : control (no phage treatment); : phage-treated
Fig. 4.Phage treatment of Japanese flounder infected with
Streptococcus iniae (Experiment 5).Fish were intrap-
eritoneally (IP) injected with phage mixture (PSiJ31,
PSiJ32, PSiJ41, PSiJ42) 12 h or 24 h after IP-injec-
tion with S. iniae strain PSi402 (See Table 3). ○ :
control (no phage treatment); : 24 h; : 12 h
Fig. 5.Kinetics of S. iniae in the Japanese flounder.Fish
were intraperitoneally injected with S. iniae strain
PSi402 at a dose of 10
5.4
CFU/fish and viable cell
counts of S. iniae in the brains and kidneys were car-
ried out using TSA plates. ○ : brain ; : kidney
188 松岡 学・橋爪貴也・神崎博幸・岩本恵美・S. C. Park・吉田照豊・中井敏博
が発生した養殖場周辺における病原体の存在はファージ
の出現(存在)に依存していると考えられる。環境に放
出された病原体が効率的にファージにより殺菌されると
したら(Imbeault et al., 2006それは水平感染による病
気の伝播に影響すると考えられるので,養殖場での病気
の消長にファージが関わっている可能性がある。
 愛媛県下のヒラメ養殖場の環境水に由来するファージ
株を使った S. iniae のファージ感受性試験では,供試し
たヒラメ病魚由来株の多くがほぼ同じファージ型に属し,
またブリやニジマス由来の菌株でもこれらのファージに
感受性を有したことから,魚類病原性 S. iniae のファー
ジ型の均質性が示唆されたしかし今回供試した
ファージ株が 株と少ないことから,本菌のファージ型6
に関しては,さらに多くのファージ株を用いてまた外国
あるいは陸上動物由来の S. iniae 株も含め詳細に検討す
る必要がある。
本研究の主眼は,ヒラメの S. iniae 感染症に対する
ファージ療法の可能性を探ることにあった。ヒラメを用
いた感染防御試験に先立って行な in vitro 増殖抑制試
験では,供試ファージは MOI = 10
–4
においても S. iniae
の増殖を一時的にではあるが抑制した。なお,培養18
間以降における増殖はファージ非感受性細胞による。ま
た,感染防御試験では,供試したヒラメのサイズ,攻撃
菌濃度,ファージの株数が異なるものの,ファージ投与
区でいずれも有意な生残率の向上が確認された。ファー
ジ投与区での試験区間の生残率に28.0%から80.0%と開
きがみられたのは,S. iniae の攻撃菌濃度の違いによると
思われるさらに魚体内の S. iniae 菌濃度が 10
7
CFU/ 尾にもなる菌攻撃24時間後にファージを接種しても
生残率が向上した。これらのことは,本菌感染症の治療
にファージ投与が有効であることを示唆している
ファージ治療試験において,感染がかなり進行したス
テージでファージを投与しても治療効果が期待できるこ
とは,アユの P. plecoglossicida 感染に対するファージ
療実験やPark et al., 2000また魚類以外でも,重篤
な状態にある VRE (バンコマイシン耐性腸球菌)感染マ
ウスに対するファージの投与実験において報告されてい
る(Biswas et al., 2002
 しかしながら,本研究でのヒラメのレンサ球菌症に対
するファージ治療においては,ファージ投与区で死亡し
た魚から,投与したファージに対して感受性がない S.
iniae 株が分離された。このことは,S. iniae では比較的
容易に in vivo で増殖可能なファージ非感受性菌,換言す
れば,病原性を有するファージ耐性菌が出現することを
示唆し,この点は S. iniae 感染症に対するファージ療法
の成否に関わる重要な問題である。病原性大腸菌の感染
に対するファージ投与の有効性がマウスあるいは産業動
物を用いた実験で報告されて以来Smith and Huggins,
1982; 1983医学分野も含めファージ療法の有効性を示
す研究報告がなされているがSulakvelidze et al., 2001;
Merril et al., 2003; Kutter and Sulakvelidze, 2004
ファージ療法の大きな問題点として,in vivo でのファー
ジ耐性菌の出現の可能性がまず指摘されてきた。しかし,
上述の大腸菌感染症に対するファージ治療では,ファー
ジ投与後に in vivo で病原性 E. coliK
+
株)が出現するこ
とは極めてまれであると報告されており(Smith and
Huggins, 1982; 1983またアユの細菌性出血性腹水症
でもファージ治療後に死亡したアユからファージ耐性 P.
plecoglossicida 菌は分離されていないPark and Nakai,
2003これらのことは,ファージ耐性菌(ファージ非
感受性変異株)は in vivo では増殖し難いか,あるいはそ
れらは主要な病原因子を欠いている可能性を示唆する。
今後は,まず in vivo で出現したファージ非感受性 S.
iniae の性状およびヒラメに対する病原性について詳細な
検討を行なうとともにS. iniae のファジ非感受性化・
耐性化機構について検討する必要がある。
謝     辞
 本研究に供試した S. iniae 菌株を分与していただいた
香川県水産試験場および抗 S. parauberis 家兎血清を供与
していただいた長崎大学金井欣也教授に深謝いたします。
本研究は,ヒラメ養殖技術高度化研究愛媛県単独事業
および先端技術を活用した農林水産研究高度化事業(課
題番号18075 によって行われた。
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... There have been a number of reports on S. agalactiae phages isolated from cow mastitis [10,11], wastewater plants [12], and mitomycin-C-induced culture of S. agalactiae clinical strains from vaginal or neonatal infection [13,14]. However, reports on phage isolation in streptococcal-infected aquaculture are far fewer, with only a few reported thus far: S. agalactiae phage HN48 from tilapia (Oreochromis niloticus) [15], Streptococcus iniae phage from olive flounder (Paralichthys olivaceus) [16], and Streptococcus parauberis phage Str-PAP-1 from olive flounder [17]. It is notable that all S. agalactiae phages isolated thus far are temperate phages, which are not preferred for phage therapy [12]. ...
... Pharmaceuticals 2023, 16, 698 ...
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The Streptococcus agalactiae outbreak in tilapia has caused huge losses in the aquaculture industry worldwide. In Malaysia, several studies have reported the isolation of S. agalactiae, but no study has reported the isolation of S. agalactiae phages from tilapia or from the culture pond. Here, the isolation of the S. agalactiae phage from infected tilapia is reported and it is named as vB_Sags-UPM1. Transmission electron micrograph (TEM) revealed that this phage showed characteristics of a Siphoviridae and it was able to kill two local S. agalactiae isolates, which were S. agalactiae smyh01 and smyh02. Whole genome sequencing (WGS) of the phage DNA showed that it contained 42,999 base pairs with 36.80% GC content. Bioinformatics analysis predicted that this phage shared an identity with the S. agalactiae S73 chromosome as well as several other strains of S. agalactiae, presumably due to prophages carried by these hosts, and it encodes integrase, which suggests that it was a temperate phage. The endolysin of vB_Sags-UPM1 termed Lys60 showed killing activity on both S. agalactiae strains with varying efficacy. The discovery of the S. agalactiae temperate phage and its antimicrobial genes could open a new window for the development of antimicrobials to treat S. agalactiae infection.
... [167][168][169][170][171][172][173] The therapeutic effects of six S. iniae lytic phages with dsDNA were studied against Streptococcosis infection in P. olivaceus at 25°C for 2 weeks ( Table 2 ). 156 Y. ruckeri is the causative bacterium of yersiniosis, known as enteric red mouth disease in freshwater salmonid fish. Yer A41, ɸ 2, ɸ 3, ɸ 3, ɸ 9 and φNC10 phages were tested as a combined or single intraperitoneal injection to treat Y. ruckeri and antibody production was reported in phage-treated fish ( Table 2). ...
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Aquaculture has grown tremendously due to the big demand for its products. However, diseases affecting aquaculture and economic losses are worldwide problems and it needs low cost, sustainable, highly efficient, specific and eco-friendly therapeutants. Due to the rising up antibiotic resistant-microorganism, bacteriophage therapy has reinvigorated to replace antibiotics in agriculture, medicine, food safety and the environment. Likewise, it also holds great promise to avoid, control and treat bacteria in aquaculture to decrease the mortality level of different aquatic animal diseases. The isolation and characterization of new phages and phage application therapy to eliminate bacterial fish and shellfish pathogens such as Vibrio , Aeromonas , Pseudomonas , Lactococcus , Yersinia , Flavobacterium , and Streptococcus was gradually reported in aquaculture literature. The present review summarizes large-scale reports in vitro or in vivo use of aquaphage studies and applications in fish diseases from the 1980s to 2022 and future directions.
Article
Streptococcus agalactiae stands out as a significant bacterial pathogen, 51 causing substantial economic losses in the aquaculture sector. Given the 52 challenges posed by multidrug resistance, this study explores the potential 53 of phage therapy as an alternative to antibiotics in biocontrol strategies. 54 The focus is on evaluating the multidrug resistance profile of S. agalactiae 55 isolated from a tilapia aquaculture farm, with particular attention to the 56 strain KSA/01, which exhibits resistance to seven structurally different 57 classes of antibiotics and a notable MAR index of around 0.6. In response to 58 this challenge, the study successfully identifies and isolates the specific and 59 lytic phage SAP-13 targeting the multidrug-resistant strain KSA/01. 60 Transmission electron microscopy reveals that SAP-13 shares morphological 61 characteristics with the Siphoviridae family. In a one-step growth curve, the 62 phage demonstrates a substantial burst size of approximately 610 PFU/cell 63 over a short burst period and the phage exhibited stability across various 64 physicochemical parameters such as temperature, pH, and salinity. In vitro 65 lytic ability of SAP-13 at different multiplicity of infection underscores its 66 potential to effectively eliminate S. agalactiae, particularly at an MOI of 67 0.01. Consequently, these findings suggest that phage SAP-13 exhibits high 68 infectivity against S. agalactiae, presenting a promising avenue for 69 addressing multidrug-resistant strains in aquaculture
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This study marks the first occasion that Streptococcus iniae has been isolated, identified, and characterized as the causative pathogen in spotted sea bass (Lateolabrax maculates). Infected fish exhibited a range of external symptoms, including scale loss, bleeding from the jaw, anus, and tail, among other signs, as well as internal manifestations such as congested liver, splenomegaly, branchial anemia, yellow fat syndrome, and intestinal edema. Notably, exophthalmia and meningoencephalitis—typical symptoms associated with previous S. iniae infections—were not observed. A predominant bacterial isolate (designated 10S01) was recovered from the pure culture of spleen of a diseased spotted sea bass in Zhuhai, China. The strain was then subjected to Gram staining, biochemical profiling, and molecular confirmation through 16S rRNA and gyrB gene, corroborating its identity as S. iniae. Pathogenicity was assessed by intraperitoneal injection challenge in spotted sea bass weighing approximately 13 g/fish, revealing a LD50 of 74 cfu/g-fish. The 10S01 strain demonstrated the ability to colonize various organs, including the spleen, liver, kidney, and brain, with a relatively higher affinity for the spleen. Furthermore, antimicrobial susceptibility testing indicated that the 10S01 strain was sensitive to 14 tested antibiotics, particularly chloramphenicol, ciprofloxacin, clarithromycin, florfenicol, ofloxacin, rifampicin, and trimethoprim/sulfamethoxazole, highlighting these as preferred treatments for S. iniae infections in spotted sea bass. These findings contribute significantly to our understanding of S. iniae pathogenesis and inform the prompt and appropriate antibiotic treatment of S. iniae infections.
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Though biosecurity and best management practices (BMPs), vaccines, immunostimulants, probiotics and prebiotics are often used for health management in aquaculture, the use of chemotherapeutics/antibiotics often becomes the preferred method of choice once the infectious disease outbreak occurs. Phage-based control of bacterial pathogens (phage therapy) has recently re-emerged as an attractive therapeutic alternative due to the global emergence of antimicrobial-resistant bacterial pathogens. Target specificity with minimal disruption of natural microbiota, auto-dosing, safety, no production of toxic metabolites/residues and relatively inexpensive production are some of the distinct advantages of phages over antibiotics. In vivo, experimental studies have demonstrated the efficacy of phage therapy through immersion, oral, injection, topical application, and anal intubation routes against Aeromonas hydrophila, A. salmonicida, Edwardsiella tarda, Flavobacterium columnare, Pseudomonas aeruginosa, Streptococcus iniae, Vibrio anguillarum, V. harveyi, V. parahaemolyticus, etc. in aquaculture. Several factors such as phage selection, therapeutic dose, age of fish, specific targeting of the pathogen, disease condition of fish, environmental conditions, and administration route influence the efficacy of phage therapy in aquaculture. The application of a mixture of phages (phage cocktail) has also been suggested to overcome the narrow host range of phages and the development of phage resistance in bacteria. At present, a few commercial phage cocktail-based products are also available for control of Yersinia ruckeri, Aeromonas spp., Pseudomonas spp., and shrimp pathogenic vibrios in aquaculture. To successfully implement phage therapy, there is a need to develop region-specific/localized phage repositories with associated safety and efficacy data which could be used to quickly formulate effective phage cocktails after the identification of pathogenic bacterial strains in specific aquaculture areas.
Chapter
Marine biotechnology is any technique that uses marine breathing creatures (or their parts) to make or modify things, or to engineer marine microbes for particular purposes. The marine environment, comprised of oceans and seas, covers more than two-thirds of the biosphere’s exterior, which inhabit over 1,400,000 species and the most ancient forms of life. Marine microorganisms are of great significance because they have changed the global climate over time and control the atmosphere. Marine organisms adapt and survive in adverse environmental conditions, making them a huge reservoir for bioactive molecules with exceptional properties and high potential. Therefore, marine environments maintain an excess of a variety of bioactive molecules with distinctive characteristics and important capabilities for biotechnological purposes. Marine reservoirs are hotspots and provide a vital natural source of healthy food and functional food components with biological properties. The organisms’ diversity in the marine environment is unknown and needs to be investigated and utilized. Modern marine biotechnology has focused on intensifying research on aquatic organisms and their secondary metabolites. Marine biotechnologists are interested in many marine organisms like crustaceans, macro and microalgae, fish and fish by-products, fungi and bacteria for healthy as well as functional food ingredients, marine drugs, and energy. Biotechnological approaches have a crucial part in exploiting marine resources for food, functional food, biomedical purposes, and bioenergy.
Chapter
One of the most underutilized biological resources in the world is the marine environment, which makes up nearly three-quarters of the Earth’s surface. A variety of organisms with unique biological systems and features can be found in the marine environment. They have evolved special characteristics that allow them to survive in a variety of hostile environments. By applying a wide variety of screening tools, extracts and purified compounds of these organisms can be studied for food processing, biological activities, and bioenergy production. Biomolecules derived from marine organisms have a wide range of applications in the food industry, including colorants, preservatives, and flavor enhancers. Some of the most useful marine-derived food ingredients are pigments, polyunsaturated fatty acids, sterols, polysaccharides, proteins, and enzymes. Among the therapeutics, more than 60 % of the active pharmaceutical formulations come from natural products or their derivatives, which have been reported to possess biological activities (anticancer, anti-inflammatory, antioxidant, antimicrobial, etc.). Using marine resources to produce biodiesel is one of the hottest areas for renewable energy. International cooperation, novel biotechnological tools, mass production of marine organisms, integration of biotechnology with other sectors, etc., will be necessary to fully explore the potential of marine sources.
Chapter
Disease outbreaks frequently impede aquaculture’s expanded expansion. Bacterial infections are one of the main issues among them. Antibiotics are frequently used in the treatment of bacterial diseases in aquaculture. Bacteriologists must create alternative control agents due to the emergence of bacteria that are resistant to standard antibiotics and bactericides as well as their possible adverse effects on the environment and human health. Therefore, new bacterial disease control methods are required. Bacteriophage therapy is thus one of the tactics. Bacteriophages, viruses that can only infect and kill highly particular types of bacteria, are potential agents with no known harmful impacts on the environment or human health. Numerous bacteriophages have been discovered to combat various fish pathogenic bacteria, and numerous studies have demonstrated how effectively they may control the spread of disease in both closed and open environments. This chapter contains details on potential bacteriophages that can fight off illnesses brought on by fish pathogenic bacteria. Bacteriophages must be bactericidal, highly specific to their host, accurately identified, free of virulence factors and stable in a variety of environmental conditions for bacteriophage therapy to be successful. With these qualities, the phage may be useful for treating vibriosis in aquaculture.
Chapter
The marine environment is a major source of biodiversity, food, energy and therapeutics. The vastness and significance of marine could be measured by the fact that about 90% of the marine environment is comprised of microbes and covers about 70% of the surface of planet Earth. Conditions like the wide range of temperature, pH, pressure and salinity further facilitate the diversification of marine organisms. Since the marine environment carries a wide range of organisms, therefore, this chapter is limited to marine bacteria only. We explained the therapeutic applications of different bacteria. Furthermore, to discover and harness the potential of marine microorganisms, we have proposed several methods and sophisticated tools that need to be developed for isolation, culturing and identification.
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The concentration of bacteriophages in natural unpolluted waters is in general believed to be low, and they have therefore been considered ecologically unimportant. Using a new method for quantitative enumeration, we have found up to 2.5 x 10(8) virus particles per millilitre in natural waters. These concentrations indicate that virus infection may be an important factor in the ecological control of planktonic micro-organisms, and that viruses might mediate genetic exchange among bacteria in natural aquatic environments.
Article
Streptococcal infections have been frequently observed in cultured freshwater fish, tilapia (Tilapia nilotica), rainbow trout (Salmo gairdneri) and ayu (Plecoglossus altivelis) at farms in various districts of Japan. The causative agents isolated from diseased tilapia, rainbow trout and ayu had the same morphological as well as biochemical characteristics. All the strains were also serological homogeneous. These strains were found to be pathogenic to freshwater fish after intraperitoneal injection. The autoclaved and hot-HCl treated cells did not react with any of the group specific sera used: Lancefield A, B, C, D, E, F, G, H, K, L, M, N, O and MG. This organism was not identical to any strains of Streptcoccus previously reported.
Article
Fish aquaculture faces important losses as a result of bacterial resistance to antibiotics. Bacteriophages have proven a useful alternative therapy in other domains, but remain to be tested with fish. The interaction between Aeromonas salmonicida HER 1107, bacteriophage HER 110, and brook trout Salvelinus fontinalis was studied in 70-L aquariums maintained at 9°C. Populations of A. salmonicida (10 colony-forming units per milliliter) declined by six log units (base 10) in 3 d when bacteriophage HER 110 was introduced in a multiplicity of infection factor of 1. Concentrations of bacteriophages and bacteria in the open water of the aquariums were 2–3 log units lower than those in gravel interstitial water. However, the relative drop in bacterial populations with time was the same in both environments. Addition of the bacteriophage HER 110 delayed by 7 d the onset of furunculosis in brook trout. Further addition of A. salmonicida HER 1107 showed that bacteriophages remained in the aquariums. Mutants of the bacterium were isolated and used as targets for bacteriophage HER 110 and nine other phages. The tests revealed that more than one phage could infect A. salmonicida HER 1107 and that mutants resistant to bacteriophage HER 110 were sensitive to one or more phages. Bacteria resistant to bacteriophage HER 110 had a slower generation time than the original strain, and the success rate of replating in tryptic soy agar (TSA) was very low. More than 25% of the mutants seemed to revert to the original-strain phenotype after a first replating in TSA. All mutants were sensitive to three or more phages. Finally, stock cultures of 10 plaque-forming units per milliliter of bacteriophage HER 110 decreased by only one log unit in 80 d when held at 4°C in liquid brain–heart infusion broth culture medium. These results suggest that bacteriophage combinations could be successfully used in preventive programs on fish farms.
Article
DESPITE the importance of cyanobacteria in global primary productivity1and of heterotrophic bacteria in the consumption of organic matter in the sea2, the causes of their mortality, particularly the cyanobacteria, are poorly understood. It is usually assumed that mortality is due to protozoan grazing3,4 rather than to viral infection, probably because abundances of phage and host in nature are presumed to be low5. Previously, either very few marine bacteriophages have been found by plaque assays6–9, or viruses have been simply observed10–12or counted13,14 by transmission electron microscopy, with the assumption that 'phage-looking' forms are locally active bacteriophages. Here we report not only high viral abundance in the ocean but also counts of bacteria and cyanobacteria in the final irreversible stage of lytic infection. The latter counts are necessary to evaluate mortality, because the sources, hosts, viability and ages of observed free viruses are unknown; even finding viruses attached to cells does not prove successful infection. Up to 7% of the heterotrophic bacteria and 5% of the cyanobacteria from diverse marine locations contained mature phage; interpretation via culture data indicates that up to 70% of the prokaryotes could be infected. These data demonstrate the existence of a significant new pathway of carbon and nitrogen cycling in marine food webs and have further implications for gene transfer between marine organisms.
Article
A mixture of two phages, B44/1 and B44/2, protected calves against a potentially lethal oral infection with an O9:K30,99 enteropathogenic strain of Escherichia coli, called B44, when given before, but not after, the onset of diarrhoea; a mixture in which phage B44/3 was replaced by phage B44/3 was effective after the onset of diarrhoea. Calves that responded to phage treatment had much lower numbers of E. coli B44 in their alimentary tract than untreated calves. Usually, high numbers of phage B44/1 and rather lower numbers of phage B44/2 or B44/3 were present in the alimentary tract of these animals. At death, most calves that had not responded to treatment with phages B44/1 and B44/2 had high numbers of mutants of E. coli B44 resistant to phage B44/1 in their small intestine. Phage-treated calves that survived E. coli infection continued to excrete phage in their faeces, at least until the numbers of E. coli B44 also excreted were low. The phages survived longer than E. coli B44 in faecal samples taken from phage-treated calves and exposed to the atmosphere in an unheated animal house. Calves inoculated orally with faecal samples from phage-treated calves that contained sufficient E. coli B44 to cause a lethal infection remained healthy. A mixture of two phages, P433/1 and P433/2, and phage P433/1 alone cured diarrhoea in piglets caused by an O20:K101,987P strain of E. coli called P433. The numbers of the infecting bacteria and phages in the alimentary tract of the piglets resembled those in the calves. Another phage given to lambs 8 h after they were infected with an O8:K85,99 enteropathogenic strain of E. coli, called S13, reduced the numbers of these organisms in the alimentary tract and had an ameliorating effect on the course of the disease. No phage-resistant mutants of E. coli S13 were isolated from the lambs. The only mutants of E. coli B44 and P433 that emerged in the calves and piglets were K30- or K101- and resistant to phage B44/1 or P433/1 respectively; those tested were much less virulent than their parent strains.
Article
Anti-K 1 phages were more active in vitro and in vivo against an 018:K1:H7 ColV+ Escherichia coli strain, designated MW, than were other phages. A single intramuscular dose of one anti-K1 phage was more effective than multiple intramuscular does of tetracycline, ampicillin, chloramphenicol, or trimethoprim plus sulphafurazole in curing mice of a potentially lethal intramuscularly or intracerebrally induced infection of MW; it was at least as effective as multiple intramuscular doses of streptomycin. When MW and the phage were inoculated into different gastrocnemius muscles of the same mice, a rapid reduction in numbers of MW organisms occurred in the MW-inoculated muscle and in other tissues; the numbers of phage particles in the MW-inoculated muscle increased rapidly and greatly. MW failed to proliferate in the brains of intracerebrally infected mice that had been inoculated intramuscularly with the phage at the same time; many more phage particles were found in the brains of these mice than in other sites. The few phage-resistant mutants of MW found in the phage-treated mich were K1-; previous studies had shown such mutants to be of greatly reduced virulence. The phage administered intramuscularly 3-5 d before challenge with a potentially lethal intramuscularly induced infection of MW was protective, the protective effect varying between phage propagated on different bacterial strains.
Article
Viruses are the most common biological agents in the sea, typically numbering ten billion per litre. They probably infect all organisms, can undergo rapid decay and replenishment, and influence many biogeochemical and ecological processes, including nutrient cycling, system respiration, particle size-distributions and sinking rates, bacterial and algal biodiversity and species distributions, algal bloom control, dimethyl sulphide formation and genetic transfer. Newly developed fluorescence and molecular techniques leave the field poised to make significant advances towards evaluating and quantifying such effects.