Role of V. splendidus related strains in an oyster mortality outbreak.. Vibrio crassostreae J5-15

In May 2011, specific pathogen free (SPF) oysters were transferred to open seawater for 2 weeks and then reintroduced in a controlled environment to reveal the disease. Mortalities started at day 3 and reached a maximum of 50% at day 5. Each day, Vibrionaceae strains infecting living oysters were isolated on selective media. The isolates were characterized by partial sequencing of a protein coding gene (gyrB) and virulence was investigated by oyster experimental infection. The superimposition of gyrB phylogeny with virulence phenotype led to the identification of a population, containing V. crassostreae type strain , and only virulent strains. To further explore the genomic diversity of strains belonging to this population 16 virulent isolates (11 from the present study and 5 from a 2001 mortality outbreak including the LGP7T type strain) and 18 non-virulent isolates were sequenced by HTS (SOM). A phylogenetic analysis of concatenated nucleic acid sequences derived from 3229 shared proteins from the 34 genome sequences suggested the clonality of some isolates within our collection This was confirmed by an ANI value >99,5% and an accessory genome <150 CDS between isolates (data not shown). Consequently only 21/34 isolate were considered as distinct strains.

2011年5月，将无特定病原体（specific pathogen free, SPF）牡蛎转移至开放海域暂养2周，随后放回受控环境以诱导疾病发生。养殖组于第3天开始出现牡蛎死亡，至第5天死亡率达到峰值50%。每日从存活牡蛎体内分离感染性弧菌科（Vibrionaceae）菌株，分离过程采用选择性培养基。通过对蛋白编码基因gyrB的部分序列进行扩增测序完成菌株分型，并通过牡蛎人工感染实验评估各分离株的毒力水平。将gyrB基因的系统发育树与毒力表型进行整合分析，最终鉴定出一个仅包含强毒株的类群，该类群包含牡蛎弧菌（V. crassostreae）模式菌株。为进一步探究该类群菌株的基因组多样性，研究人员采用高通量测序（High-throughput sequencing, HTS，SOM）对16株强毒株（本研究分离得到11株，另有5株源自2001年牡蛎死亡暴发事件，其中包含LGP7T模式菌株）以及18株非强毒株进行了全基因组测序。基于34个全基因组序列中3229个共有蛋白的串联核酸序列开展系统发育分析，结果显示本研究收集的部分分离株具有克隆同源性；该结论通过平均核苷酸一致性（Average Nucleotide Identity, ANI）值>99.5%以及菌株间附属基因组差异小于150个编码序列（Coding Sequence, CDS）得到了验证（相关数据未展示）。因此，最终仅将34株分离株中的21株认定为独立菌株。