芽孢杆菌V4和胶红酵母对鲑鳟鱼生长免疫及肠道菌群影响研究

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	芽孢杆菌V4和胶红酵母对鲑鳟鱼生长免疫及肠道菌群影响研究
	王纯
学位类型	博士
导师	刘鹰
	2017-05-20
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	海洋生态学
关键词	鲑鳟鱼芽孢杆菌v4 胶红酵母免疫肠道微生物
摘要	鲑鳟鱼是世界性的主养冷水鱼类，其肌肉富含人体必需的氨基酸、不饱和脂肪酸等，是优质的高端水产品，市场需求巨大。工厂化循环水养殖是实现鲑鳟鱼高密度、高品质、高产量的重要生产方式之一，目前，高密度循环水条件下的鲑鳟鱼养殖出现了诸如鱼类病害多发等问题，制约着鲑鳟鱼养殖业的快速可持续发展。亟需从加强鱼体健康的角度出发，探究出一条生态安全可行的鲑鳟鱼疾病预防策略，在此情形下，筛选和研制高效的益生型菌株，应用于鲑鳟鱼工厂化养殖过程，是改善鱼类健康、增强鱼类抵抗病原菌能力的优先策略。本研究以大西洋鲑和虹鳟为研究对象，将筛选研制的具有特殊拮抗活性的潜在益生型菌株应用于鲑鳟鱼工厂化养殖，探究其对鲑鳟鱼的益生效果，从鱼类生长，存活，免疫、拮抗病原菌能力，肠道微生物菌群结构等多角度进行综合评价，为后续商业化推广和应用提供坚实的理论基础。本研究的主要成果如下： 1. 通过采集健康及患病大西洋鲑鱼，初步构建了感染杀鲑气单胞菌的鲑鳟鱼致病模型，联合运用DGGE和16S rRNA基因测序技术，深入探究鲑鳟鱼肠道内微生物菌群结构，以期查明鱼类健康状态与肠道微生物菌群之间的关系，研究发现：健康大西洋鲑肠道中的主要微生物组成为变形菌门（Proteobacteria，44.33%），放线菌门（Actinobacteria，17.89%），拟杆菌门（Bacteroidetes，15.25%）和厚壁菌门（Firmicutes，9.11%），其中，Oxalobacteraceae科和微球菌科（Micrococcaceae）的某些属，鞘氨醇单胞菌属（Sphingomonas），链霉菌属（Streptomyces），乳球菌属（Lactococcus）和Pedobacter菌属的细菌是健康大西洋鲑肠道中的优势菌属。然而病鱼肠道内优势微生物门类则稍有差异，主要为变形菌门（Proteobacteria，70.46%），拟杆菌门（Bacteroidetes，7.59%），厚壁菌门（Firmicutes，7.55%）和绿弯菌门（Chloroflexi，2.71%），气单胞菌科（Aeromonadaceae）和腐螺旋菌科（Saprospiraceae）的某些属，Aliivibrio菌属，弧菌属（Vibrio），和梭菌属（Clostridium）是病死鱼肠道中的优势菌属，研究证实健康大西洋鲑与病鱼肠道中微生物菌群结构存在显著差异。基于实验室严格控制条件下所构建的精准大西洋鲑致病菌侵染模型研究发现，杀鲑气单胞菌侵染急剧降低了大西洋鲑肠道中微生物菌群种类，且随着攻毒浓度的升高逐步下降，同时造成大西洋鲑肠道微生态平衡的严重破坏，显著降低大西洋鲑肠道微生物菌群多样性，鱼类肠道微生物菌群与鱼类健康状态之间关系紧密。 2. 验证了筛选的芽孢杆菌V4菌株和胶红酵母复合菌剂对工厂化循环水养殖虹鳟的益生效果。分别发酵芽孢杆菌V4（B. velezensis V4）和胶红酵母（R. mucilaginosa）制备复合菌剂，添加投喂虹鳟，探究其对于虹鳟幼鱼生长、存活、饵料利用等方面影响，同时综合运用PCR-DGGE指纹图谱分析及基于16S rRNA基因的高通量测序技术研究复合菌剂对虹鳟胃及肠道黏膜微生物、肠道内容物中微生物菌群影响，研究发现：T1菌剂添加组（B. velezensis V4/ R. mucilaginosa，5 × 10⁶/ 5 × 10⁷ CFU/g）虹鳟的增重、特定生长率均显著提升，饵料系数显著降低，而所有菌剂添加组虹鳟的存活率均显著改善。基于虹鳟肠道微生物菌群结构的UPGMA和PCoA分析显示，对照组与复合菌剂添加组肠道中微生物菌群结构并未显著差异，但复合菌剂添加组微生物菌群多样性（Chao1指数，Shannon多样性指数等）均有所提高，益生型嗜冷杆菌属（Psychrobacter），芽孢乳杆菌属（Sporolactobacillus），乳球菌属（Lactococcus）及链霉菌属（Streptomyces）在复合菌剂添加组得到有效提升，同时，潜在致病菌伯克氏菌属（Burkholderia）的丰度得到有效遏制。 3. 芽孢杆菌V4（B. velezensis V4）及胶红酵母（R. mucilaginosa）应用于大西洋鲑幼鱼养殖，实验的研究发现：添加复合菌剂能够显著促进大西洋鲑生长，提高存活率，提高饲料利用率，在养殖初始实验阶段，复合菌剂添加组大西洋鲑血清中丙二醛（MDA），谷丙转氨酶（GPT）和一氧化氮（NO）的含量显著降低（P < 0.05），同时，随着复合菌添加浓度的升高，血清中MDA、GPT和NO的含量持续降低。在T1菌剂添加组中（B. velezensis V4 / R. mucilaginosa，5 × 10⁶ CFU g^–1/ 5 × 10⁷ CFU g^–1），血清中总超氧化物歧化酶（t-SOD）、谷胱甘肽（GSH）、谷胱甘肽-过氧化物酶（GSH-Px）、过氧化氢酶（CAT）、酸性磷酸酶（ACP）、总抗氧化力（T-AOC）和免疫球蛋白M（IgM）的含量显著高于对照组（P < 0.05）。皮质醇（cortisol）的含量在所有复合菌添加组中均有显著提高（P < 0.05）。复合菌剂添加实验组攻毒后的累积死亡率低于对照组，表现出一定的优越性。复合菌剂显著提高了大西洋鲑幼鱼在杀鲑气单胞菌侵染后血清中总超氧化物歧化酶（t-SOD）和过氧化氢酶（CAT）活力，同样，谷草转氨酶（GOT），谷丙转氨酶（GPT）和酸性磷酸酶（ACP）的活性在复合菌添加组有显著升高，尤其在感染后的48小时。溶菌酶活性（LZM）在T1和T3实验组中有部分提升，血清免疫球蛋白M（IgM）和皮质醇（cortisol）的水平在复合菌剂添加组有显著提升，说明复合菌的使用能有效调控大西洋鲑鱼的免疫应答，尤其在杀鲑气单胞菌侵染后复合菌投喂组大西洋鲑表现出强烈的益生作用，同时对于大西洋鲑肠道微生物菌群微生态结构无显著影响。 4. 评估了单一芽孢杆菌V4菌株对鲑鳟鱼类的影响。经过70天的养殖实验及后续的杀鲑气单胞菌侵染实验，共同探究和验证了芽孢杆菌V4菌株对虹鳟的生长、存活、饲料利用、免疫能力、拮抗病原菌及肠道菌群的影响，综合结果发现芽孢杆菌V4添加能够提高虹鳟存活率，提高了饲料利用率，同时增强了虹鳟的免疫应答、抗氧化应激及拮抗病原菌能力，尤其在杀鲑气单胞菌侵染后芽孢杆菌V4投喂组虹鳟表现出强烈的益生作用，同时芽孢杆菌添加组，蜡样芽胞杆菌（Bacillus cereus）丰度有升高趋势，甲基营养型芽孢杆菌（Bacillus methylotrophicus）和鼠乳杆菌（Lactobacillus murinus）的丰度也随着芽孢杆菌V4添加浓度的增大而升高，且甲基营养型芽孢杆菌的增长趋势显著，综合结果显示芽孢杆菌V4对虹鳟肠道微生物菌群微生态结构无显著影响，未影响其肠道内原有菌群的微生态平衡。
其他摘要	Salmonids aquaculture industry has become the mainstay of cold water fish in markets worldwide. The salmonids are rich in essential amino acid and unsaturated fatty acid, which can effectively reduce the cholesterol in the blood, so as to prevent from cardiovascular disease actively caused by arteriosclerosis. Salmonids are high-quality aquatic products which are very popular. Recirculting aquaculture of salmonids is the most promising way to meet the market requirement. RAS was characterized by high density, high quality and low pollution. Aquaculture environmental degradation, water pollution, fish disease and many other issues restrict the rapid development of salmonids aquaculture. Efficient disease prevention strategies were urgent. Strain screening of probiotic bacteria was considered to be a promosing way to improve the fish health, enhance the ability of resistance to pathogens, which can be a sustainable ecological priority strategy of salmonids aquaculture. The present study was to assess the effects of a potential probiotic Bacillus velezensis V4 and Rhodotorula mucilaginosa on growth, immune response, antioxidant capability, disease resistance and intestinal microbiota of juvenile salmonids reared in a recirculating aquaculture system. This studies provide strong theoretical basis for commercialization and application of this bacteria. The main results are summarized as follows: 1. In order to understand the differences in the intestinal microbiota of Atlantic salmon with different health statuses, we sampled the intestinal content of healthy and unhealthy Atlantic salmon (Salmo salar L.), the ambient water of unhealthy fish, and the biofilter material in the recirculating aquaculture system (RAS). Denatured gradient gel electrophoresis (DGGE) was used to scan the intestinal mictobiota of fish intestine. The V4–V5 regions of the prokaryotic 16S rRNA genes in the samples were further analyzed by MiSeq high-throughput sequencing. Representative members of the intestinal microbiota were identified. The intestinal microbiota of the healthy fish included Proteobacteria (44.33%), Actinobacteria (17.89%), Bacteroidetes (15.25%), and Firmicutes (9.11%), among which the families Micrococcaceae and Oxalobacteraceae and genera Sphingomonas, Streptomyces, Pedobacter, Janthinobacterium, Burkholderia, and Balneimonas were most abundant. Proteobacteria (70.46%), Bacteroidetes (7.59%), and Firmicutes (7.55%) dominated the microbiota of unhealthy fish, and Chloroflexi (2.71%), and Aliivibrio and Vibrio as well as genera in the family Aeromonadaceae were most strongly represented. Overall, the intestinal hindgut microbiota differed between healthy and unhealthy fish. Much more accurate diseased fish model was built to study the relationship between healthy and diseased fish infected by Aeromonas salmonicida. The species of intestinal microbitota were significantly decreased by the infection of A. salmonicida, and also the intestinal bacterial diversity. Serious microecological imbalance in intestinal microbiota was caused by the A. salmonicida infection. 2. To assess the effects of probiotic compound of Bacillus velezensis V4 CGMCC 10149 and Rhodotorula mucilaginosa CGMCC 1013 on the growth performance and gut microbiota of rainbow trout (Oncorhynchus mykiss). Rainbow trout were reared in recirculating aquaculture system (RAS) and fed with four experimental diets which contained compound probiotics of 0/0 CFU g^-1(Control, B. velezensis V4/ R. mucilaginosa), 5 × 10⁶/ 5 × 10⁷ CFU/g (T1), 1.5 × 10⁷/1.5 × 10⁸CFU/g (T3), 2.5 × 10⁷ / 2.5 × 10⁸CFU/g (T5) for 40 days, respectively. Weight gain ratio, feed conversion ratio and specific growth rate were significantly enhanced in diet T1 at the end of the trial. Survival rate was significantly improved in the probiotics treatments, especially in diet T3. The gut microbiota of the rainbow trout was assessed by MiSeq sequencing of the V4 region of 16S rRNA genes. Interestingly, no obvious deviations were observed in the whole gut microbial community structures of the different treatments according to unweighted pair-group method with arithmetic mean (UPGMA) and principal coordinate analysis (PCoA). Compared with the control, the mean Chao1 estimator, Shannon-Wiener index, and Simpson index were higher in the probiotic compound treatments. In the gut of rainbow trout fed with the probiotic compound diets, the relative abundance of probiotic Psychrobacter, Sporolactobacillus, Lactococcus and Streptomyces were increased, while the potential pathogens such as Burkholderia were lower than those of control. 3. A feeding trial of 62 days was conducted to investigate the effects of dietary administration of a probiotic compound consisting of B. velezensis V4 and R. mucilaginosa on growth, immune response, antioxidant capability, disease resistance and intestinal microbiota of juvenile Atlantic salmon (Salmo salar L.) reared in a recirculating aquaculture system (RAS). Administration of the probiotic compound had significant effects on growth performance and feed utilization. Mortality was also significantly reduced (P < 0.05). The level of malondialdehyde (MDA), GPT (glutamic pyruvic transaminase) and NO (nitric oxide) was decreased by the addition of probiotic compound in feeding trial. T-SOD (total superoxide dismutase), GSH (glutathione), GSH-Px (glutathione peroxidase), CAT (catalase), ACP (acid phosphatase), T-AOC (total antioxidant capacity) and IgM was enhanced (P < 0.05) in T1 group. Cumulative mortality was also reduced in the probiotic diets. The t-SOD, CAT, GOT, GPT and ACP were increased by the infection of A. salmonicida challenge, especially 48 h after challenge. Immune Lysozyme, IgM and cortisol was also elevated. This results demonstrated B. velezensis V4 and R. mucilaginosa compound as a beneficial dietary probiotics for juvenile Atlantic salmon reared in a RAS with the capability of improving growth performance and enhancing disease resistance by immunomodulation. No obvious deviations were observed in the whole gut microbial community structures of the different treatments. 4. Furthermore, a 70-day systematic laboratory experiment was designed to explore the effect of dietary administration of single B. velezensis V4 on growth, survival rate, immune response, antioxidant capability, disease resistance and intestinal microbiota of juvenile salmonids. The results represented that survival rate and feed conversion ratio was elevated by the addition of B. velezensis V4. No obvious weight gain was found. Strong immune response, antioxidant capability and disease resistance was found, especially after the challenge of A. salmonicida. The micro-ecological balance of intestinal microbiota of rainbow trout was less affected. The relative abundance of Bacillus cereus, Bacillus methylotrophicus and Lactobacillus murinus were accelerated by the addition of B. velezensis V4, especially for Bacillus methylotrophicus.
学科领域	生物学 ; 微生物学 ; 微生物生态学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/136608
专题	实验海洋生物学重点实验室
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	王纯. 芽孢杆菌V4和胶红酵母对鲑鳟鱼生长免疫及肠道菌群影响研究[D]. 北京. 中国科学院大学,2017.

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