弧菌攻毒模型及凡纳滨对虾肠道对哈氏弧菌侵染的生理、免疫反应研究

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	弧菌攻毒模型及凡纳滨对虾肠道对哈氏弧菌侵染的生理、免疫反应研究
	夏青
学位类型	硕士
导师	王雷
	2015-05-05
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	水产养殖
关键词	弧菌攻毒模型 Cox比例风险模型肠道损伤组织切片免疫基因凡纳滨对虾
摘要	弧菌广泛分布于海水养殖环境内，在水产养殖中，弧菌一直被认为是很多海洋鱼类或无脊椎动物特别是虾的主要致病菌。凡纳滨对虾是世界对虾养殖的主要经济品种，随着对虾养殖规模的快速发展，养殖环境日益恶化，由弧菌引发的对虾疾病，影响对虾的生长繁殖，引起对虾的大量死亡，已经在全球范围内造成了重大经济损失，成为对虾养殖产业发展的主要制约因素。解决病害问题需要从病原感染机理等研究入手。本研究共分为两个部分：第一部分基于Cox比例风险模型，探究哈氏弧菌不同菌株、剂量及感染方式对凡纳滨对虾的攻毒效果的影响。目前相当多的研究揭示了许多因素影响弧菌病的爆发，其疾病动态发展机制亟需探讨。很多学者针对弧菌与对虾死亡的相互作用展开研究，这对如何有效控制攻毒试验的风险因素极为重要。在本研究中，Cox比例风险模型首次应用于水产动物疾病学研究，有效分析了在弧菌的攻毒试验中对虾动态生存情况，并定量描述了弧菌风险因素的影响。预后指数的建立也为今后攻毒试验选择条件提供了指导。主要结果如下： 1.采取log-rank检验方法对不同弧菌攻毒浓度、不同侵染途径及不同弧菌菌株作用下的对虾生存期进行比较，高浓度弧菌攻毒(10⁸cfu/mL)较低浓度弧菌攻毒(10⁷cfu/mL)，在更短时间内引起了对虾持续大量的死亡现象(P = 0.038)；注射攻毒比浸浴攻毒能够更有效地引发弧菌感染(P < 0.05)；相比菌株Vp，菌株Vh对对虾生存期缩短有更大的影响(P = 0.042)。 2.注射攻毒导致对虾死亡的风险是浸浴攻毒的40.4倍(HR=40.4; P＝0.000)。高浓度弧菌攻毒组(10⁸cfu/mL)对低浓度弧菌攻毒组(10⁷cfu/mL)死亡风险比为5.9(HR=5.9, P < 0.000)。Vh毒力较强，是Vp毒力的4.8倍(HR＝4.8, P < 0.000)。 3. 在对虾攻毒试验中，侵染方式因素对对虾生存时间影响最大( β=3.698, P < 0.05)，攻毒剂量因素次之(β=1.778, P < 0.05)，弧菌菌株因素影响最小(β=1.561, P < 0.05). 第二部分研究针对凡纳滨对虾肠道对哈氏弧菌浸浴的组织病理变化及转录水平的免疫应答。通过组织切片探究对虾肠道组织形态在不同浓度哈氏弧菌(低浓度组1.05×10⁵cfu/mL，中浓度组1.05×10⁶cfu/mL及高浓度组1.05×10⁷cfu/mL)的浸浴攻毒作用下的动态变化，采用实时荧光定量PCR测定抗脂多糖因子基因(ALF)、对虾素基因(Pen-4c)和Crustin基因(Cru)以及溶菌酶基因(LZM)、脂肪酸结合蛋白基因(Fabp)等免疫相关基因的表达水平变化，并对肠道及水体中的弧菌数目及分布情况进行监测。主要结果如下： 1.浸浴攻毒是弧菌侵染对虾肠道并引发感染的有效方式。对虾肠道的感染程度可分为轻微感染及重度感染。轻度感染主要发生于低中浓度组的中后期及高浓度组侵染早期，上皮细胞形态发生改变，黏膜层开始与固有层分离。重度感染主要发生于低中浓度组后期及高浓度组中期，上皮细胞完全溃散并脱落，肌层也发生损伤。随着弧菌浓度的提高和攻毒时间的延长，中肠组织损伤情况逐渐加重。攻毒后40h内，对虾肠道组织形态持续恶化，未见组织修复。 2.肠道受到哈氏弧菌侵染后8-16h，ALF、Pen、Cru及LZM表达量能够迅速上调，32h后均回落至初始水平(P<0.05)。Cru在哈氏弧菌侵染作用下表达量变化最大。Fabp基因32h内各攻毒剂量组表达情况未有极大波动，基本较对照组维持在2-6倍的表达水平，并且总体上并无显著提高(P<0.05)。 3. 攻毒后，三种攻毒浓度组水体中弧菌均处下降趋势，未出现弧菌大规模增殖的现象。在32h后，低、中、高浓度组无显著性差异并普遍趋于稳定；低、中、高攻毒组中，对虾肠道弧菌在0-8h通过水浴攻毒方式经口传播大量进入对虾肠道，8h-24h维持稳定，16h后肠道弧菌小量外排至32h菌数回落。
其他摘要	Vibrio has been widely recognized as a primary pathogen of many commercially cultured invertebrate species the world over. Shrimp culture problems associated with Vibrio has a direct effect on production, feed efficiency, and growth performance, which may limit shrimp culture development. Litopenaeus vannamei is very important in shrimp culture. With the rapid development of shrimp culture, aquaculture environment has been deteriorating, and vibriosis has become the main factors to limit the development of shrimp-culture industry. This study was divided into two parts, The first part based on the Cox proportional hazard model evaluated the effects of bacterial dosage, infection route and Vibrio strain in experimental challenges of Litopenaeus vannamei. While considerable research has revealed many important factors affecting outbreaks of vibriosis, many additional aspects of the dynamics of associated mechanisms need to be explored. A greater understanding of the interaction between Vibrio and shrimp mortality has been achieved, which should prove to be helpful when attempting to control the risk factors associated with the experimental challenge. In this work, Cox model was first used to analyze Vibrio hazards in aquaculture and it was the first model to capture the dynamics shrimp survivals under Vibrio experimental challenges. For this purpose a new numerical method was developed, and to study the quantitative contribution of seemingly-obvious risk factors that lead to shrimp vibriosis. A prognostic index (PI), constructed from the Cox model, would be of great assistance in evaluating and choosing experimental treatments. Results indicate that： 1.Based on Kaplan–Meier estimates of Vibrio dosage, infection route, and Vibrio strain, we were able to construct survival curves that display the survival distribution of shrimps in challenge tests. Survival curves were compared by means of applying the log-rank test. The infection event resulted in continuous and extensive mortality of shrimps in high dose group (10⁸cfu/mL) than low dose group (10⁷cfu/mL, P = 0.038); infection rates following injection were significantly different from those following immersion (P < 0.05); Vh had a significantly higher virulence in this test than the case for Vp (P = 0.042). 2.Injection was found to be the more efficient method for infecting shrimp (HR=40.4; P＝0.000), which means that the mortality risk is 40.4 times greater than that obtained following immersion. Relative to the low dose, of 10⁷cfu/shrimp, the high dose group, of 10⁸cfu/shrimp, was regarded as having a higher hazard risk in terms of causing shrimp mortality (HR=5.9, P < 0.000). The virulence of Vh was found to be 4.8 times that of Vp(P < 0.000). 3. In the experimental challenge for estimating shrimp survival, the infection route had the higher effect on mortality (β=3.698, P < 0.05), followed by dose (β=1.778, P < 0.05) and Vibrio strain (β=1.561, P < 0.05). The second part studied on the pathological changes and transcriptional response to the immersion infection by different doses Vibrio harveyi in shrimp Litopenaeus vannamei mid-gut. The dynamic changes in intestinal morphology were explored. Changes in expression of anti-lipopolysaccharide factor gene (ALF), penaeidin gene (Pen-4c), crustin gene (Cru), lysozyme gene (LZM) and fatty acid binding protein gene (Fabp) were quantified by qPT-PCR, while the number of Vibrio in intestine and water was monitored. The results list following, 1.Immersion is an efficient method in terms of Vibrio colonization and intestinal infections. The intestinal infections could be divided into two levels: mild infection and intense infection. The mild infection occurred in middle/terminal phase of low dose group and middle dose group. It also occurred in early phase of high dose group. In mild infection, the intestinal epithelial cells appeared degradation and the mucosal folds occurred shrinkage and separation. The intense infection occurred in terminal phase of low dose group and middle dose group. It was also common in middle phase of high dose group. And then, the intestinal epithelial cells appeared complete collapsing and escaping, and the muscular layer was also damaged. As the dose of Vibrio increased and infection time added, the intestinal tissue damaged severely. Within 40h post infection, shrimp gut morphology deteriorated continuously, and the tissue was not repaired. 2.At 8-16h after infection by V. harveyi, the expression levels of ALF, Pen-4c, Cru and LZM up-regulated immediately and were back to the initial level at the end of 32h. Cru has the biggest increase, while the expression level of Fabp did not significantly increase (P<0.05). 3.The number of vibrio in water of three treatments was decreasing post infection, and no multiplication occurred. At the end of 32h, the population of vibrio in water was stable. In three treatments, the cumulative bacteria uptake was increasing in intestine via immersion at 0-8h and balanced at 8-24h.
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/22780
专题	海洋生物技术研发中心
作者单位	中国科学院海洋研究所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	夏青. 弧菌攻毒模型及凡纳滨对虾肠道对哈氏弧菌侵染的生理、免疫反应研究[D]. 北京. 中国科学院大学,2015.

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