凡纳滨对虾细菌性红体病病原的分子特征与耐药性

Molecular characteristics and antimicrobial sensitivity of bacterial pathogen from the outbreak of Litopenaeus vannamei red-body disease

为探明引起凡纳滨对虾细菌性红体病的病原,从病虾肝胰脏分离得到10株优势菌,经回归感染实验证实其为引起此次红体病的病原菌。Vitek与16S rRNA序列分析显示,分离株均为副溶血弧菌。基于dnaE-gyrB-recA-dtdS-pntA-pyrC-tnaA的多位点序列分型(multilocus sequencetyping,MLST)表明,这些菌株形成3个新序列型(ST),其中1株为ST413,7株为ST414,2株为ST415;ST413包含新等位基因型recA-166与tnaA-121,ST414则含有新等位基因型gyrB-219。MLST结果提示,这些副溶血弧菌分离株并非来自单一克隆,呈现出一定水平的分子多样性。但这些菌株均含有大流行群(PG)的分子标记toxRS与VPA1168,并具有相同的毒力基因构成(tlh+tdh-trh-T3SS1+T3SS2-)与耐药谱,其tdh与trh的缺失并未影响细菌对凡纳滨对虾的致病力。综上所述,引起此次凡纳滨对虾红体病的10株副溶血弧菌可能为PG的不同变异株。

Red-body disease is one of the most severe diseases of Litopenaeus vannamei.An outbreak of L.vannamei red-body disease happened in a large-scale breeding farm in Zhejiang Province,2011,with the mortality rate of 90%.A total of ten bacterial isolates were collected from the hepatopancreas of diseased shrimps,which were responsible for this outbreak.These isolates were identified as Vibrio parahaemolyticus by Vitek and 16S rRNA sequence analysis.Multilocus sequence typing（MLST） based on the concatenated genes dnaE-gyrB-recA-dtdS-pntA-pyrC-tnaA demonstrated that these isolates belonged to three novel sequence types（ST）,with one isolate to ST413,seven to ST414 and two to ST415.ST413 contained two novel allelic profiles,recA-166 and tnaA-121,and ST414 harbored one novel allelic profile,gyrB-219.These novel allelic profiles and STs had been confirmed and deposited by the MLST website（http：//pubmlst.org/vparahaemolyticus/）.MLST results indicated these V.parahaemolyticus isolates did not originate from the same clone and exhibited remarkable genetic diversity.On the other hand,all of these isolates contained molecular markers for pandemic group,including a unique sequence within the toxRS operon,encoding transmembrane proteins involved in the regulation of virulence-associated genes,and VPA1168 within an 16-kb insertion,which encodes a hypothetical protein with approximately 80% similarity to the Mn2＋ and Fe2＋ transporter in V.vulnificus.Also these isolates had the same virulence-associated gene composition（tlh＋tdh-trh-T3SS1＋T3SS2-） and antimicrobial sensitivity profiling.Absence of tdh and trh,which had traditionally been thought to be critical for the virulence of V.parahaemolyticus,did not lead to the reduction of bacterial pathogenicity in L.vannamei.Overall,these V.parahaemolyticus isolates might represent distinct variants within PG.