lsolation and ldentification of the Pathogen Causing Skin Ulcer Disease in Cynoglossus semilaevis Günther and lts Sensitivity to Chinese Herbal Medicine

Guisheng GAO，Yanying ZHANG，Qiumei SHl*，Guangping GAO，Hongsheng HAN，Xiaoyuan HUANG，Baofeng JlAO，Yaoyao CHANG

1.Key Laboratory of Preventive Veterinary Medicine of Hebei Province/College of Animal Science and Technology，Hebei Normal University of Science&Technology，Qinhuangdao 066600，China；

2.Qinhuangdao Liangfeng Marine Ecological Science and Technology Co.，Ltd.，Qinhuangdao 066600，China

lsolation and ldentification of the Pathogen Causing Skin Ulcer Disease in Cynoglossus semilaevis Günther and lts Sensitivity to Chinese Herbal Medicine

Guisheng GAO1，Yanying ZHANG1，Qiumei SHl1*，Guangping GAO1，Hongsheng HAN2，Xiaoyuan HUANG2，Baofeng JlAO2，Yaoyao CHANG1

1.Key Laboratory of Preventive Veterinary Medicine of Hebei Province/College of Animal Science and Technology，Hebei Normal University of Science&Technology，Qinhuangdao 066600，China；

2.Qinhuangdao Liangfeng Marine Ecological Science and Technology Co.，Ltd.，Qinhuangdao 066600，China

In this study，the pathogen causing skin ulcer disease in Cynoglossus semilaevis Günther was isolated for morphological observation，physiological and bio-chemical identification.According to the result，the isolated pathogen was identified as Vibrio harveyi.The results ofrecurrentinfection ofC.semilaevis Günther showed that the pathogen was strongly pathogenic to C.semilaevis Günther.In or-der to explore the pathogenesis，outer membrane protein （OMP）gene of C.semilaevis Günther was detected by PCR.The results showed that all the three repre-sentative strains harbored OMP gene.According to the results of sensitivity test of the pathogen to Chinese herbal medicine，Galla Chinensis，F(xiàn)ructus Mume，F(xiàn)ructus Hippophae and Lignum Sappan exerted strong antibacterial effects against V.harveyi；Pericarpium Granatiexhibited slightantibacterialeffectagainstV.harveyi；Pericarpium Citri Reticulatae，Rhizoma Acori Graminei，Herba Houttuyniae，Herba Portulacae，Herba Andrographis，Eucalyptus globulus Labill.and Herba Menthae Heplocalycis had little effect on V.harveyi.Galla Chinensis，F(xiàn)ructus Mume，F(xiàn)ructus Hippophae and Lignum Sappan were prepared into three prescriptions，among which prescription 1（Galla Chinensis+Fructus Mume）exhibited the strongest antibacterial effect.

Cynoglossus semilaevis Günther；Skin ulcer disease；Pathogen；Outer membrane protein；Chinese herbal medicine

I n recent years，coastal industrial aquaculture is rapidly developing，but the benefit of the aquaculture industry has been greatly affected by disease epidemics.In previous stud-ies，common bacterial diseases and their epidemiological characteristics in a circulating water aquaculture plant of Cynoglossus semilaevis Günther in Qinhuangdao City were investigated. The results show that skin ulcer is the most common bacterial disease in cir culating water aquaculture of C.semilaevis Günther，which poses a serious threat to the reproductive survival rate. At present，few systematic studies have been reported on diseases of C.semilaevis Günther，but Little is available on occurrence and patho-genesis of these diseases.In this study，skin ulcer disease in aquacul-ture of C.semilaevis Günther was in-vestigated systematically，aiming at providing basis for further prevention and control of skin ulcer disease.

Clinical Symptoms and PathologicalChanges in Cynoglossus semilaevis Günther infected with Skin Ulcer Disease

In April 2015，skin ulcer disease occurs in a coastal industrial aquacul-ture plant of C.semilaevis Günther in Qinhuangdao City.C. semilaevis Günther infected with skin ulcer dis-ease exhibited skin ulcers，fin base swelling，fin end ulcers，local gill fila-ment edema with telangiectasia and congestion， liver and pancreas swelling，renal tissue degeneration，necrosis and ulceration，red blood cell deposition and hemoglobin precipita-tion in spleen，gallbladder enlargement with telangiectasia，and large amounts of catarrhal mucus in intestines（Fig.1，F(xiàn)ig.2）.

Pathogen lsolation

Liver，pancreas，kidney tissues and ascites of C.semilaevis Günther infected with skin ulcer disease were collected，inoculated to a nutrient agar plate，and incubated at 28℃for 24 h. Morphological observation results show that round translucent，pale yel-low or light brown bacterial colonies were grown with smooth and moist surface and neat edges （Fig.3）.The colonies were inoculated to a blood a-gar plate and incubated at 28℃ for 12-24 h.As shown in Fig.4，complete-ly transparentzones ofhemolysis formed on the plate.As shown in Fig.5，the pathogen was a Gram-negative bacterium with blunt round ends，and individual cells were slightly curved.

Physiological and Bio-chemicalldentification of the Pathogen

Three predominant strains were isolated from liver，pancreas，kidney tissues and ascites of C.semilaevis Günther infected with skin ulcer dis-ease to detect the physiological and biochemical characteristics （Table 1）. According to the results，these strains were positive to lipase，arginine dihy-drolase，D-trehalose，D-maltose and β-galactosidase but negative to D-glu-cose，L-arabitol，D-mannitol，malonate，D-arabinitol，L-aspartate arylamidase，β-glucosidase，ornithine decarboxy-lase，5-ketogluconate and lysine de-carboxylase，indicating that culture，physiological and biochemical charac-teristics of the isolated strains were consistent with Vibrio harveyi.There-fore，the pathogen causing skin ulcer disease in C.semilaevis Günther was identified as V.harveyi.

Recurrent lnfection of C.semilaevis Günther with the Pathogen

A strain isolated from liver and pancreas tissues was incubated with LB broth and inoculated to five healthy C.semilaevis Günther.A C.semilaevis Günther was used as the control，and other four individuals were inject-ed with 0.3 ml of bacterial liquid（con-taining 2×106colonies/ml）.At 12 h post-infection，experimental fish swam slowly with lassitude，gradual skin ul-ceration and loss of appetite，which died successively within 72 h，but the controlfish exhibited no abnormal performance.Autopsy results showed gill congestion，organ adhesion and liver congestion，with similar symp-toms to naturally infected C.semilaevis Günther.Strains were isolated from dead C.semilaevis Günther for culture and identification.According to the re-sults，the original injection bacterium exhibited consistent morphological and biochemical characteristics with V.harveyi，indicating that the original injection bacterium was the pathogen causing skin ulcer disease in C.semilaevis Günther.

Detection of Outer Mem-brane Protein Gene（OMP）of the Pathogen

Genomic DNA of V.harveyi was extracted in accordance with the intro-ductions of genomic DNA extraction kit（Fig.6）.The results showed that ge-nomic DNA of three V.harveyi strains was successfully extracted.Using ge-nomic DNA of V.harveyi as a tem-plate，933 bp specific band were am-plified by PCR，which was consistent with the expected size （Fig.7），indicat-ing that outer membrane protein gene（OMP）of V.harveyi was successfully amplified and detected.

The amplified OMP gene of V.harveyi was sequenced and sub-mitted to GenBank.By using BLAST software，four reference strains pub-lished in GenBank were obtained for comparison and analysis of nucleotide homology.The results showed that OMP gene sequences of three V.harveyi strains shared 98.7%-100%ho-mologies with reference strains.To be specific，OMP gene sequence of rep-resentative strain 3 shared 100%ho-mology with reference strain JQ946883；OMP gene sequences of three representative strains shared 98.8%-99.4%homologies with each other（Fig.8）.

Table 1 Biochemical identification results

Sensitivity Test of the Pathogen to Chinese herbal medicine

As shown in Fig.9 and Table 2，among 12 single herbs used in this study，Galla Chinensis，F(xiàn)ructus Mume，F(xiàn)ructus Hippophae and Lignum Sappan exerted strong antibacterial effects against V.harveyi，among which Gal-la Chinensis had the highest antibac-terial activity；Pericarpium Granati ex-erted no significant antibacterial effect against V.harveyi；Pericarpium Citri Reticulatae，Rhizoma Acori Graminei，Herba Houttuyniae，Herba Portulacae，Herba Andrographis，Eucalyptus globulus Labill.and Herba Menthae Hep-localycis had little effect on V.harveyi.

According to the results of sensi-tivity test of the pathogen to 12 single herbs， Galla Chinensis， Fructus Mume， Fructus Hippophae and Lignum Sappan，which exerted the strongest antibacterial effects，were prepared into prescription 1 （Galla Chinensis+Fructus Mume），prescrip-tion 2（Galla Chinensis+Fructus Hip-pophae）and prescription 3（Galla Chinensis+Lignum Sappan）for sensitivi-ty test.The results showed that pre-scription 1 （Galla Chinensis+Fructus Mume）exhibited the strongest an-tibacterial effect.

Table 2 Antibacterial circle diametermm

Discussions

In the current aquaculture pro-duction，bacterial diseases are still the most important diseases endangering cultured fishes［1-4］.According to the dis-ease records from Key Laboratory of Preventive Veterinary Medicine of Hebei Province and existing research-es，bacterial diseases of C.semilaevis Günther are mainly caused by Vibrio，and most pathogens belong to Gram-negative bacteria，such as V.harveyi，V.splendidus and V.anguillarum.Vib-rio strains causing other flatfish dis-eases can also cause bacterial dis-eases in C.semilaevis Günther to some extent.In this study，V.harveyi is the main pathogen causing skin ul-cer disease in C.semilaevis Günther. Statistics show that V.harveyi is the most important pathogen that causes diseases of maricultural animals［5］，which can induce ulceration symptoms in Scophthalmus maximus［6］，F(xiàn)ugu obscurus［7］，Pseudosciaena crocea and other maricultural species，cause de-generation and necrosis of internal organs of diseased fishes［8-9］，lead to physiological metabolic disorders of diseased fishes［10］and reduce their im-munity and resistance to diseases，thereby causing the death of diseased individuals.In the present study，the pathogen causing skin ulcer disease in C.semilaevis Günther was identified as V.harveyi；all the three representa-tive strains harbored OMP gene.Ac-cording to the results of sensitivity test of the pathogen to Chinese herbal medicine，V.harveyiwas strongly pathogenic to C.semilaevis Günther. Galla Chinensis， Fructus Mume，F(xiàn)ructus Hippophae and Lignum Sappan exerted strong antibacterial effects againstV.harveyi；prescription 1（Galla Chinensis+Fructus Mume）ex-hibited the strongest antibacterial ef-fect.

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Responsible editor：Xiaohui FAN

Responsible proofreader：Xiaoyan WU

半滑舌鰨腐皮病病原分離鑒定及對中草藥的敏感性實驗

高桂生1，張艷英1，史秋梅1*，高光平1，韓紅升2，黃曉媛2，繳寶豐2，?，幀?（1.河北省預防獸醫(yī)學重點實驗室，河北科技師范學院動物科技學院，河北秦皇島 066600；2.秦皇島糧豐海洋生態(tài)科技股份有限公司，河北秦皇島066600）

對引起半滑舌鰨腐皮病的病原進行了形態(tài)學觀察、生理生化鑒定，結(jié)果為哈維氏弧菌，動物回歸試驗顯示該菌對半滑舌鰨有較強的致病作用。為了探索其致病機理，采用PCR方法進行了外膜蛋白（OMP）基因檢測，結(jié)果發(fā)現(xiàn)，3株代表菌均攜帶OMP基因。通過中草藥藥敏試驗的結(jié)果顯示：五倍子、烏梅、沙棘、蘇木四種單味中藥對哈維氏弧菌有較強的抑菌作用；陳皮、石菖蒲、魚腥草、馬齒莧、穿心蓮、桉樹葉、薄荷等對病原哈維氏弧菌無作用；根據(jù)中藥組方法則擬定了三個組方，其中由五倍子和烏梅組成的組方抑菌效果最強。

半滑舌鰨；腐皮??；病原；外膜蛋白；中草藥

河北省科技廳獎勵性后補助項目（15926620H）；秦皇島市科技局科技支撐計劃（201401A067，201402B043）。

高桂生（1964-），男，河北玉田人，副教授，碩士，從事水產(chǎn)養(yǎng)殖方面研究，E-mail：Ggs1964@163.com。*通訊作者。

2015-08-28

Supported by Post-award Grants Program of Science and Technology Department of HebeiProvince （15926620H）；Scienceand TechnologySupportProgram of Qinhuangdao MunicipalBureau of Science and Technology （201401A067，201402B043）

*Corresponding author.E-mail：Ggs1964@163.com

Received：August 28，2015 Accepted：October 8，2015

修回日期 2015-10-08