大菱鲆脾腎結(jié)節(jié)病病原菌的分離和鑒定*

李 杰 劉耀寬,3 白 露,2 陳四清,2 閻永偉 莫照蘭,2

大菱鲆脾腎結(jié)節(jié)病病原菌的分離和鑒定*

李 杰1劉耀寬1,3白 露1,2陳四清1,2閻永偉1莫照蘭1,2①

(1. 中國(guó)水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所 農(nóng)業(yè)農(nóng)村部海水養(yǎng)殖病害防治重點(diǎn)實(shí)驗(yàn)室 青島海洋科學(xué)與技術(shù)試點(diǎn)國(guó)家實(shí)驗(yàn)室海洋漁業(yè)科學(xué)與食物產(chǎn)出過程功能實(shí)驗(yàn)室 青島 266071；2. 上海海洋大學(xué)水產(chǎn)與生命學(xué)院 上海 201306；3. 青島農(nóng)業(yè)大學(xué)海洋科學(xué)與工程學(xué)院 青島 266109)

2017年夏季，江蘇省連云港市某養(yǎng)殖場(chǎng)大菱鲆出現(xiàn)大規(guī)模死亡，發(fā)病魚體表無明顯癥狀，解剖可見脾臟、腎臟出現(xiàn)白色散在結(jié)節(jié)，從發(fā)病魚的內(nèi)臟中分離得到1株優(yōu)勢(shì)菌SM-Myco001。人工感染實(shí)驗(yàn)結(jié)果顯示，SM-Myco001可以引起大菱鲆內(nèi)臟結(jié)節(jié)癥狀并造成魚類死亡，且在高養(yǎng)殖水溫(22℃)條件下發(fā)病更為劇烈。生理生化鑒定和16S rRNA基因分析結(jié)果顯示，SM-Myco001屬于海分枝桿菌()。本研究首次報(bào)道了我國(guó)養(yǎng)殖大菱鲆感染海分枝桿菌的病例，可為大菱鲆養(yǎng)殖過程中的疾病防控提供參考。

大菱鲆；海分枝桿菌；分枝桿菌病；結(jié)節(jié)

大菱鲆()是我國(guó)北方海水工廠化養(yǎng)殖的主要品種之一，根據(jù)《2017年中國(guó)漁業(yè)統(tǒng)計(jì)年鑒》，我國(guó)鲆鰈類養(yǎng)殖產(chǎn)量合計(jì)131389 t，大菱鲆產(chǎn)量居首位。我國(guó)的大菱鲆養(yǎng)殖業(yè)經(jīng)過20多年的發(fā)展，養(yǎng)殖成本不斷降低，從高檔酒店逐步進(jìn)入大眾市場(chǎng)，為人民生活水平的提高做出了貢獻(xiàn)，但大菱鲆產(chǎn)業(yè)的發(fā)展也遇到了瓶頸。病害是影響大菱鲆養(yǎng)殖業(yè)的重要因素，不但引起魚類死亡，造成直接經(jīng)濟(jì)損失，還可能導(dǎo)致藥物濫用、藥殘超標(biāo)，造成消費(fèi)者恐慌，進(jìn)而影響市場(chǎng)價(jià)格。對(duì)大菱鲆的病害已有較多的研究，目前已報(bào)道多種大菱鲆病原，包括鰻弧菌() (鄒玉霞等, 2004)、哈維氏弧菌() (范文輝等, 2005)、溶藻弧菌() (張偉妮等, 2006)、遲緩愛德華氏菌() (李筠等, 2006)、殺鮭氣單胞菌() (呂俊超等, 2009)、虹彩病毒(史成銀等, 2005)和盾纖毛蟲等(王印庚等, 2004)，這些流行病學(xué)的研究為大菱鲆養(yǎng)殖疾病防控提供了理論依據(jù)。

2017年，本實(shí)驗(yàn)室從江蘇省連云港市某養(yǎng)殖場(chǎng)患脾腎結(jié)節(jié)病的大菱鲆中分離到1株優(yōu)勢(shì)菌，鑒定為海分枝桿菌()，通過人工感染實(shí)驗(yàn)證明該菌株對(duì)大菱鲆的致病性，并對(duì)其發(fā)病溫度進(jìn)行初步分析，以期為我國(guó)養(yǎng)殖大菱鲆的病害防控提供參考。

1 材料與方法

1.1 病魚來源

2017年7~9月，江蘇省連云港市某養(yǎng)殖場(chǎng)工廠化養(yǎng)殖大菱鲆出現(xiàn)大量死亡事件。發(fā)病時(shí)，養(yǎng)殖水溫為18℃~22℃，發(fā)病魚體重為500~650 g，共養(yǎng)殖大菱鲆4萬尾左右，發(fā)病魚約為10000尾，其中約死亡5000尾。應(yīng)用廣譜抗生素進(jìn)行治療效果不明顯，待水溫下降至18℃以下，大菱鲆逐漸恢復(fù)正常。

1.2 細(xì)菌分離

取發(fā)病魚的鰓、體表粘液和尾鰭，分別在顯微鏡下觀察，檢測(cè)寄生蟲感染情況。取發(fā)病魚的脾臟、腎臟組織分別在2216E和Middlebrook 7H10 (含10% OADC，青島海博)固體培養(yǎng)基平板劃線進(jìn)行病原菌分離，28℃培養(yǎng)4周后，將優(yōu)勢(shì)菌落進(jìn)行純化培養(yǎng)，純化培養(yǎng)后的細(xì)菌用甘油保存于-80℃冰箱備用。

1.3 人工感染實(shí)驗(yàn)

將細(xì)菌接種于酸性羅氏斜面培養(yǎng)基(青島海博)，28℃培養(yǎng)4周后，用PBS沖洗菌體，離心收集并重懸。健康大菱鲆購自煙臺(tái)一大菱鲆養(yǎng)殖場(chǎng)，體重為100 g左右，實(shí)驗(yàn)前隨機(jī)選取5%的大菱鲆進(jìn)行解剖觀察和內(nèi)臟勻漿涂布，確定大菱鲆未攜帶病原。將大菱鲆隨機(jī)分為2組，每組20尾，飼養(yǎng)水溫分別為16℃和22℃，每尾大菱鲆腹腔注射0.1 ml 0.5×107CFU/ml的菌液，設(shè)置對(duì)照組飼養(yǎng)于相同條件下，并注射等體積的PBS。注射感染后，正常飼養(yǎng)觀察30 d，解剖和記錄實(shí)驗(yàn)魚死亡情況并進(jìn)行病原分離。實(shí)驗(yàn)結(jié)束后，將實(shí)驗(yàn)組和對(duì)照組剩余的大菱鲆麻醉致死，解剖觀察記錄內(nèi)臟結(jié)節(jié)情況。

1.4 細(xì)菌鑒定

采用引物27F(5￠-AGAGTTTGATCCTGGCTCAG- 3￠)和1492R (5￠-TACGGCTACCTTGTTACGACTT-3￠)對(duì)細(xì)菌的16S rRNA基因進(jìn)行擴(kuò)增并測(cè)序(Lane, 1991)，獲得的序列信息在GenBank和EzBioCloud進(jìn)行同源性比對(duì)，用MEGA 5.0軟件采用鄰位相連法(Neighbor- Joining)構(gòu)建系統(tǒng)發(fā)育進(jìn)化樹；根據(jù)Chiodini(1986)的方法，檢測(cè)細(xì)菌的Tween-80水解、尿素酶、68℃下過氧化氫酶、硫酸酯酶、硝酸還原酶、色素產(chǎn)生、5% NaCl和麥康凱瓊脂(MacConkey Agar)生長(zhǎng)等生理生化指標(biāo)。

2 結(jié)果

2.1 病魚癥狀

發(fā)病魚體表無明顯癥狀，偶見白便。解剖可見脾臟、腎臟出現(xiàn)白色散在結(jié)節(jié)，直徑為0.5~3 mm，腸道發(fā)紅，有白色內(nèi)容物(圖1)。顯微鏡下未觀察到寄生蟲感染，利用Middlebrook 7H10培養(yǎng)基，從發(fā)病魚的肝臟、脾臟和腎臟可以分離到大量形態(tài)一致的白色不透明圓形菌落，選擇單菌株進(jìn)行純化后將其命名為SM-Myco001。將平板放置于可見光下12 h，菌落變?yōu)辄S色。從2216E平板上未分離到明顯優(yōu)勢(shì)菌。

圖1 發(fā)病大菱鲆癥狀

2.2 人工感染實(shí)驗(yàn)

人工感染實(shí)驗(yàn)結(jié)果如表1所示，在16℃水溫養(yǎng)殖條件下，25%的感染魚出現(xiàn)死亡，解剖可見明顯脾腎結(jié)節(jié)癥狀，30 d后有結(jié)節(jié)癥狀的魚合計(jì)占35%；當(dāng)養(yǎng)殖水溫為22℃時(shí)，65%的感染魚出現(xiàn)死亡，解剖可見明顯脾腎結(jié)節(jié)癥狀，30 d后有結(jié)節(jié)癥狀的魚合計(jì)占90%。利用Middlebrook 7H10培養(yǎng)基，從結(jié)節(jié)組織中均可分離到與SM-Myco001菌落形態(tài)一致的優(yōu)勢(shì)菌。2組對(duì)照組實(shí)驗(yàn)魚均未發(fā)生死亡或出現(xiàn)結(jié)節(jié)癥狀。

表1 SM-Myco001對(duì)大菱鲆人工感染實(shí)驗(yàn)

Tab.1 Experimental infection of S. maximus infected with SM-Myco001

2.3 病原菌鑒定

PCR擴(kuò)增菌株SM-Myco001的16S rRNA基因序列(MH636826)，測(cè)序結(jié)果經(jīng)比對(duì)分析顯示，在GenBank中同源性>99%的前15株菌均為海分枝桿菌()，EzBioCloud比對(duì)結(jié)果也顯示其為海分枝桿菌。從NCBI數(shù)據(jù)庫中選擇不同分枝桿菌屬()的細(xì)菌，使用MEGA 5.0構(gòu)建系統(tǒng)進(jìn)化樹。結(jié)果顯示，SM-Myco001與海分枝桿菌和潰瘍分枝桿菌()聚為一支(圖2)。

生理生化檢測(cè)結(jié)果顯示，SM-Myco001在Tween-80水解、尿素酶、68℃下過氧化氫酶、硫酸酯酶和色素產(chǎn)生等反應(yīng)為陽性，硝酸還原酶、5% NaCl和麥康凱瓊脂生長(zhǎng)為陰性，與海分枝桿菌的生理生化指標(biāo)相符。由于潰瘍分枝桿菌Tween-80水解和硫酸酯酶反應(yīng)為陰性，綜合基因序列進(jìn)化分析和生理生化結(jié)果，將病原菌SM-Myco001鑒定為海分枝桿菌。

圖2 基于16S rRNA基因序列構(gòu)建的系統(tǒng)發(fā)育進(jìn)化樹

3 討論

分枝桿菌是廣泛存在于水體環(huán)境中的條件致病菌，龜分枝桿菌()、偶發(fā)分枝桿菌()和海分枝桿菌等多種分枝桿菌屬的細(xì)菌引起野生或養(yǎng)殖魚結(jié)核病，在世界各地均有報(bào)道(Frerichs, 1993; Belas,1995)。由于培養(yǎng)條件苛刻，生長(zhǎng)周期較長(zhǎng)，分枝桿菌感染的情況經(jīng)常被忽略。本研究從患脾腎結(jié)節(jié)病的大菱鲆中分離得到優(yōu)勢(shì)菌SM-Myco001，經(jīng)生理生化檢測(cè)和16S rRNA基因分析，確定為海分枝桿菌。人工感染實(shí)驗(yàn)結(jié)果顯示，SM-Myco001可以引起大菱鲆內(nèi)臟結(jié)節(jié)癥狀，并導(dǎo)致感染魚的死亡。當(dāng)水溫由16℃升高至22℃后，感染魚的死亡率和出現(xiàn)結(jié)節(jié)的比例都有所提高，這與自然發(fā)病時(shí)的條件較為吻合，在夏季高溫期間發(fā)病，水溫降低后大菱鲆逐漸恢復(fù)正常。大菱鲆最適養(yǎng)殖溫度在20℃以下，高溫會(huì)影響大菱鲆的生長(zhǎng)狀態(tài)和免疫力，增加條件致病菌的感染幾率，同時(shí)，分枝桿菌的生長(zhǎng)速度也會(huì)加快，這可能是高溫增加大菱鲆結(jié)核病發(fā)病率的原因。

分枝桿菌可以通過水體、養(yǎng)殖器械、餌料等多種渠道進(jìn)行水平傳播(Hedrick, 1987; Jacobs, 2009)，在魚體狀態(tài)不佳或受到環(huán)境脅迫條件下發(fā)作(Zanoni, 2008)。除消毒和隔離外，目前尚無有效預(yù)防和治療魚類分枝桿菌感染的方法(Jacobs, 2009)。海分枝桿菌屬于人魚共患病原，也可以感染人類，引起游泳池肉芽腫(Fish tank granuloma)(Lewis, 2003)，因此，有效的預(yù)防和應(yīng)對(duì)海水養(yǎng)殖魚類海分枝桿菌感染，對(duì)養(yǎng)殖魚類、養(yǎng)殖從業(yè)者和消費(fèi)者的安全都具有重要意義。

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(編輯 馬璀艷)

Isolation and Identification ofAssociated with Splenic and Renal Granuloma Disease of Cultured Turbot ()

LI Jie1, LIU Yaokuan1,3, BAI Lu1,2, CHEN Siqing1,2, YAN Yongwei1, MO Zhaolan1,2①

(1. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266071; 2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306; 3. Marine Science and Engineering College, Qingdao Agricultural University, Qingdao 266109)

With the highest annual production among flatfish, turbot () is reared as the major industrial aquaculture marine fish species in North China. In the culture process, members of this species are subject to infection with a variety of pathogens. In 2017, massive death of reared turbot occurred in a farm in Lianyungang City, Jiangsu Province. Externally, no clinical signs were observed for most of the diseased fish. However, after dissection, splenic and renal tubercles were found in all the diseased fish. Homogeneous colonies were isolated from diseased or moribund fish and were named as SM-Myco001. Healthy turbot were subjected to challenge tests by intraperitoneal injection using SM-Myco001. SM-Myco001 was found capable of causing death in turbot, especially at a higher water temperature (22℃). The diseased turbot displayed clinical signs, such as splenic and renal tubercles, similar to those observed in naturally infected fish. SM-Myco001 was identified asbased on bacterial morphology, analytical profile index identification, and 16S rRNA gene sequencing. This study suggests that a strain of.is the causal agent of splenic and renal tubercle disease in turbot. As a first report, to our knowledge, this study provides a new perspective on disease control in the flat fish aquaculture industry in China.

;; Mycobacteriosis; Granuloma

MO Zhaolan, E-mail: mozl@ysfri.ac.cn

* 國(guó)家重點(diǎn)研發(fā)計(jì)劃(2018YFC0311300)、國(guó)家自然科學(xué)基金-山東省人民政府聯(lián)合基金(U1706205)、中國(guó)水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所基本科研業(yè)務(wù)費(fèi)(20603022017008)和鰲山科技創(chuàng)新計(jì)劃(2015ASKJ02)共同資助[This work was supported by National Key R&D Program of China(2018YFC0311300), NFSC-Shandong Joint Fund (U1706205), the Central Public-Interest Scientific Institution Basal Research Fund, YSFRI, CAFS (20603022017008), and Aoshan Technology Innovation Program (2015ASKJ02) ]. 李 杰，E-mail: lijie@ysfri.ac.cn

莫照蘭，研究員，E-mail: mozl@ysfri.ac.cn

2018-07-21,

2018-09-03

S943

A

2095-9869(2019)05-0195-05

10.19663/j.issn2095-9869.20180721001

http://www.yykxjz.cn/

李杰, 劉耀寬, 白露, 陳四清, 閻永偉, 莫照蘭. 大菱鲆脾腎結(jié)節(jié)病病原菌的分離和鑒定. 漁業(yè)科學(xué)進(jìn)展, 2019, 40(5): 195–199

Li J, Liu YK, Bai L, Chen SQ, Yan YW, Mo ZL. Isolation and identification ofassociated with splenic and renal granuloma disease of cultured turbot (). Progress in Fishery Sciences, 2019, 40(5): 195–199