对虾肝胰腺坏死症微生态机理研究—从相关性到因果关系

Shrimp (Litopenaeus vannamei) hepatopancreas necrosis syndrome (HPNS) is characterized of wide range of morbidity and high mortality rate, while its pathogenesis is currently unclear. Based on available literatures and our previous works, we hypothesize that deteriorative water quality induces the virulence of opportunistic pathogens, stresses shrimp and disrupts the gut microbiota, which cumulatively facilitates the invasion of pathogens, thereby resulting in disease outbreak. This proposal will integrate Illumina sequencing and PathoChip techniques to track the dynamics of gut bacterial community over shrimp development stages and HPNS progression. Based on the microecological equilibrium theory, we will explore the interplay among water quality, toxic algae, bacterioplankton communities, virulence genes, shrimp gut microbiotas and HPNS severity, thereby identifying key virulence genes that facilitate the pathogenicity of pathogens and the underlying driving environmental factors. Candidate pathogens would be primarily identified via multiple key virulence genes, which are isolated by using the predicted media given their 16S rDNA gene sequences. Further, we validate the causal pathogens by stimulating the external conditions and re-introduced infections. Tracing the source to track the dispersal route of causal pathogens from rearing water to shrimp, and uncover how the pathogens overcome the barriers imposed by the shrimp gut commensals and immunity. Towards above findings, the pathogenesis of shrimp HPNS will be illustrated from a microecological perspective. After ruling out the baseline of gut bacterial virulence genes as shrimp aged, we intend to identify disease-discriminatory virulence genes that characteristic of shrimp HPNS. Further, we attempt to establish a strategy that employs the relative abundances of disease-discriminatory virulence genes (assayed by qPCR) serve as independent variables, for quantitatively predicting the incidences of shrimp HPNS.

凡纳滨对虾（Litopenaeus vannamei）肝胰腺坏死症发病范围广、致死率高，其致病机理不明。基于文献和前期工作，我们推测水质恶化诱导条件病原菌致病性、胁迫并改变对虾肠道菌群，最终病原菌成功定殖导致病害。本项目拟结合Illumina测序和病原菌芯片（PathoChip）技术监测对虾养殖和肝胰腺坏死症病害过程中肠道菌群和毒力基因组成。基于微生态平衡理论探究水质/有毒藻-浮游细菌-毒力基因-肠道菌群-病害严重程度间相互关系。网络互作分析鉴定关键的致病毒力基因及环境诱因；多毒力基因确定潜在病原菌，预测其培养基配方选择性分离。模拟发病环境和多病原菌侵染时序特征，回复侵染验证。追根溯源揭示病原菌从水体到对虾肠道中的传播规律、如何突破宿主免疫和肠道共生菌的定殖拮抗；探究肝胰腺坏死症的微生态学机理。同时，尝试建立以qPCR检测的病害指示毒力基因相对丰度为自变量，定量预测对虾病害风险的技术。

世界动物卫生组织已将急性肝胰腺坏死病（AHPND）列为制约虾养殖业的七种最具传染性的细菌性疾病之一。AHPND因发病急、致死率高和感染广泛，成为影响虾养殖业的最具致病性和破坏性的疾病。因此，亟需明确其致病源及发病机理，并建立定量预测技术实现轻病早治。本研究从患AHPND对虾中分离获得一株携带pirB毒力基因的鳗弧菌，回复侵染，表型和肠道中定殖等因果关系验证为AHPND的致病源。发现水温> 27.7℃, DO < 7.6, pH >8.12, NO3- >2.08 mg/L可能导致病害的暴发。增温促进鳗弧菌编码鞭毛相关基因表达，富营养化上调转录因子和VI型分泌系统的表达。对虾通过TLRs和NLRs激活下游NF-kB响应病原菌侵染，而病原菌能够通过抑制对虾MAPK途径，逃逸免疫清除导致病害。去除13个生长阶段区分菌种和23个特定疾病的区分菌种后，我们鉴定到18个对虾病害共性指示ASVs，利用其相对丰度及权重为自变量，总准确率为90.0%。上述优化步骤大大提高了诊断模型的性能（未优化62.6%对优化90.0%）。无论病因，其相对丰度在健康和相应的病虾间一致性增减，表明它们对虾的健康状况的一致性响应。为了能够在水产中应用，我们进一步在宏基因分析的基础上，运用相同优化步骤筛选到4个毒力基因：VF0460 EF-Tu，VF0072 ClpC，VF0082 IV pili和VF0542 LPS。仅4个毒力基因就提供了最高的准确率，总准确率为88.2%。发表致谢SCI论文15篇，授权发明专利2项，计算机软件著作权4件，培养研究生6名；项目成果获2021年度宁波市科学技术进步奖 1项。技术在对虾养殖场中试3年，降低病害风险18.2%，增收3706万元。

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