浙江省主要养殖区凡纳滨对虾(Litopenaeusvannamei)红体病病原研究

Study on pathogens from outbreaks of Pacific white shrimp Litopenaeus vannamei red-body disease in main cultured area of Zhejiang Province.

对2012—2013年浙江省凡纳滨对虾(Litopenaeus vannamei)主要养殖区的红体病虾进行病原鉴定及其分子特征与耐药性研究,并与2011年的结果进行比较分析.结果表明:通过Vitek、16SrRNA序列分析与病毒特异性聚合酶链反应,2012—2013年在红体病虾中同时检测出副溶血弧菌(Vibrio parahaemolyticus)与传染性皮下和造血组织坏死病毒(infectious hypodermal and haematopoietic necrosis virus,IHHNV),而2011年在红体病虾中仅检测出副溶血弧菌,未检测出IHHNV;3年均未检测出桃拉综合征病毒(Taura syndrome virus,TSV)和白斑综合征病毒(white spot syndrome virus,WSSV).基于dnaE-gyrB-recA-dtdSpntA-pyrC-tnaA的多位点序列分型结果表明,副溶血弧菌分离株具有较高水平的分子多样性:2011—2013年各年的分离株均分属多个序列型(sequence type,ST),来自位于进化树不同亚枝、亲缘关系较远的不同克隆;2012—2013年的分离株经历了较高水平的分子变异,其中ST918与ST919为首次报道的副溶血弧菌新ST;2011年分离株与2012—2013年分离株的等位基因和序列型特征具有较大差异,ST414与ST114分别代表2011年与2012—2013年红体病的优势ST.2011—2013年的副溶血弧菌分离株均属于大流行群,并具有相同的毒力基因谱(tlh+tdh-trh-T3SS1+T3SS2-),tdh与trh的缺失并未影响细菌的致病力.分离株对抗菌药物尤其是氨基糖苷类的耐药性呈现加重趋势.说明本次凡纳滨对虾红体病可能是多种致病因子共同作用的结果,亟须建立红体病病原监测体系,并探明细菌性病原与病毒性病原混合感染对致病性的影响及其互作机制,以提出综合防控方案.

Summary Pacific white shrimp ( Litopenaeus vannamei) is one of the most important commercially cultured aquaculture species around the world . Zhejiang Province represents as one of the main cultured areas in China . In recent years , concurrent with the rapid expanding and intensifying of aquaculture , infectious diseases in Pacific white shrimp L . vannamei have been steadily increasing . Red‐body disease is one of the most common and severe diseases of Pacific white shrimp . From 2011 to 2013 , recurrent outbreaks of Pacific white shrimp red‐body disease occurred in large‐scale breeding farms within the main cultured area of Zhejiang Province , which caused severe economic losses to the shrimp culture industry . Affected shrimps showed typical signs of red bodies , irregular black spots , listless swimming on water surface and reduced feed activities , accompanying with mass mortalities .This study was conducted to investigate the bacterial and viral pathogens from 2012 - 2013 outbreaks and to illuminate their molecular characteristics and antimicrobial sensitivities , which were compared with those from 2011 outbreak . Using Vitek biochemical test , 16S rRNA sequence analysis and virus specific polymerase chain reaction , V ibrio parahaemolyticus and infectious hypodermal and haematopoietic necrosis virus ( IHHNV ) were simultaneously identified from the diseased Pacific white shrimps of 2012 - 2013 outbreaks , but except V . parahaemolyticus , IHHNV was not detected from the diseased ones of 2011 outbreak ; meanwhile , Taura syndrome virus ( TSV) and white spot syndrome virus ( WSSV) were not detected from the diseased Pacific white shrimps of 2011 - 2013 outbreaks . Although V . parahaemolyticus was positive from all outbreaks during three years , these isolates exhibited remarkable genetic diversity by multilocus sequence typing ( MLST ) based on the concatenated genes dnaE‐gyrB‐recA‐dtdS‐pntA‐pyrC‐tnaA . The isolates from the same year belonged to multiple sequence types ( STs) , originated from distinct clones located on separate sub ‐branches , and the isolates from 2012 - 2013 outbreaks underwent high level of molecular variation with distinct allelic profiles and STs as compared with from 2011 outbreak . ST414 and ST114 represented dominant STs from 2011 outbreak and 2012 -2013 outbreaks , respectively . Novel STs , ST 918 ( registration No . id‐1353) and ST919 ( registration No . id‐1354) , had been confirmed and deposited in the PubMLST database . All of these isolates from 2011 - 2013 outbreaks belonged to the pandemic group , which was responsible for the majority of clinical cases since 1996 , determined by specific molecular markers , i . e . , a unique sequence within the toxRS operon encoding transmembrane proteins involved in the regulation of virulence‐associated genes , and V PA1168 within an 16‐kb insertion which encoded a hypothetical protein with approximate 80% similarity to the Mn2 + and Fe2 + transporter . Also , these isolates displayed the same virulence‐associated gene profile , containing thermolabile hemolysin ( encoded by tlh) and type Ⅲ secretion systems 1 ( T3SS1) but lacking thermostable direct hemolysin ( encoded by tdh) , TDH‐related hemolysin ( encoded by trh) and type Ⅲ secretion systems 2 ( T3SS2) . These results revealed that these isolates were atypical virulent isolates ( tdh+ and/or trh+ ) or atypical pandemic group isolates ( mostly tdh+ trh‐ ) . Absence of tdh and trh , which had traditionally been thought to be critical for the virulence of V ibrio , did not lead to the reduction of bacterial pathogenicity . Moreover , these isolates also showed elevated level of resistance against antimicrobials , especially aminoglycoside .In conclusion , Pacific white shrimp red‐body disease might be caused by multiple infections with bacterial and viral pathogens . It is necessary to develop the surveillance system of pathogens implicated in red‐body disease , and to clarify the pathogenic mechanism of polymicrobial infection to establish comprehensive prevention and control schemes .