病例研究：未知病因的凡纳滨对虾溞状幼体的病原和微生物组分析

Case Studies: Pathogenic Agent and Microbiome Analysis for Zoea of Litopenaeus vannamei Suffering from an Unknown Disease

中文关键词: 病例研究  凡纳滨对虾  溞状幼体  未知病因  菌群多样性

英文关键词: Case study  Litopenaeus vannamei  Zoea  Unknown disease  Microbial diversity

中文摘要:

      从某对虾育苗场2个育苗池中分别采集了发生摄食下降、活力降低及死亡率增高等症状的未知疾病的凡纳滨对虾(Litopenaeus vannamei)溞状幼体3期(Z3)和1期(Z1)的样品。用PCR检测白斑综合征病毒(WSSV)、传染性皮下及造血组织坏死病病毒(IHHNV)、急性肝胰腺坏死病副溶血弧菌(VpAHPND)、桃拉综合征病毒(TSV)、传染性肌坏死病毒(IMNV)、黄头病毒(YHV)、虾肝肠胞虫(EHP)、偷死野田村病毒(CMNV)和虾血细胞虹彩病毒(SHIV)等9种已知病原为阴性。组织病理学诊断观察到肝胰腺小管上皮细胞内存在不明褐色团块。使用2216E培养基对致病菌分离鉴定，得到2株溶藻弧菌(Vibrio alginolyticus)，浸浴感染悉生卤虫(Artemia franciscana)幼体后的平均存活率分别为58%和83%。采用Illumina HiSeq高通量测序方法对这2个溞状幼体样品中细菌16S rRNA基因的2个高变区(V3~V4)进行总细菌菌群的测序，分析揭示了未知病因病虾中细菌菌群的多样性和相对丰度，门水平上2个样品的优势菌群均包括变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)，但相对丰度有显著差异；在属水平上相对丰度最高的均为弧菌属(Vibrio)，在2个样品中的丰度分别为74.3%和60.5%，此外，Z1样品相对高丰度(21.9%)的属为黏着杆菌属(Tenacibaculum)。育苗管理信息及上述病例分析结果提示，该疾病可能是由于在过期存放的幼体饲料质量下降所致的营养障碍情况下，有一定致病性的溶藻弧菌条件性感染所引起的疾病。

英文摘要:

      A shrimp hatchery of Litopenaeus vannamei was infected by an unknown disease during the zoea stage with declined feeding, reduced activity, and increased mortality. In order to diagnose and explore the pathogenic factors causing the zoea disease, we collected samples from two diseased rearing ponds at zoea stage 3 (Z3) and zoea stage 1 (Z1), respectively. PCR detection for nine known pathogens, including WSSV (White spot syndrome virus), IHHNV (Infectious hypodermal and hematopoietic necrosis virus), VpAHPND (Acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus), TSV (Taura syndrome virus), IMNV (Infectious myonecrosis virus), YHV (Yellow head virus), EHP (Microsporidian Enterocytozoon hepatopenaei), CMNV (Covert mortality nodavirus), and SHIV (Shrimp hemocyte iridescent virus) showed negative results. Histopathological diagnosis showed unknown brown particles in the epithelial cells of the hepatopancreatic tubules. The results of bacterial isolation and identification for potential pathogens resulted in only two isolates of Vibrio alginolyticus on 2216E media. The average survival rate of gnotobiotic brine shrimp (Artemia franciscana) nauplii immersion- challenged with these isolates were 58% and 83% respectively. The microbial communities were profiled by high-throughput sequencing of the V3~V4 hypervariable region of 16S rRNA gene using the Illumina HiSeq sequencing platform. Meta-16S-sequencing revealed the microbial diversities and their relative abundance in the zoea stage of L. vannamei. The results showed that Proteobacteria, Bacteroidetes, and Firmicutes were the most dominant phyla in the entire bacterial community from both samples, but their relative abundance was obviously different. At the genus level, Vibrio was the most abundant genus in both samples, at 74.3% and 60.5%, respectively, and the relative abundance of Tenacibaculum (21.9%) was the second highest genus in the Z1 stage sample. Collectively, the information regarding the management of larval rearing and the above results of the case study imply that the disease may be caused by an opportunistic infection with some strains of V. alginolyticus having moderate pathogenicity under the dystrophia of larva caused by quality degradation of larval feed due to the expiration of storage.