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Infection and defense mechanisms in micro-injuries on the body surface of fish.

鱼类体表微损伤的感染与防御机制。

基本信息

批准号：
11460087
负责人：
WAKABAYASHI Hisatsugu
金额：
$ 6.72万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11460087/
关键词：
Body surface Micro-injuries Pseudomonas plecoglossicica Glugea plecoglossi Quantitative PCR in situ hybridization Uvitex 2B Green fluorescent protein labeling Flavobacterium psychrophilum Thelohanellus hovorkai Glugea plecolossi in s

项目摘要

1) Distribution of Pseudomonas plecoglossicida infection in the tissues of immersion challenged ayu (Plecoglossis altivelis) was studied by real-time quantitative polymerase chain reaction (Q-PCR). Fish were immersed in 10^7 CFU/mL of P.plecoglossicida suspension for 15 min. The fish were sampled at 1, 3, 6, 12, 24, 48, 72h post-infection (p.i.) and the skin, gill, liver, spleen and kidney, and the blood were tested by Q-PCR.The skin and/or gill was likely portal entry for the bacterium since P.plecoglossicida was found in 1 and 3 h p.i., respectively. However, the bacterium was more concentrated in liver, spleen and kidney at 6 h p.i. These results indicate that P.plecoglossicida can successfully invade ayu within 6 h p.i. Septicemia occurred at 48 h p.i., because high concentrations of the target DNA were suddenly found in the blood of infected fish.2) A green fluorescent protein (GFP) gene was placed in a shuttle vector plasmid of pME4150 and GFP vector plasmids were construction. T … More he P.plecoglossicida carring pSKTO3 gave the highest expression of GFP, and was stable under non-selective condition. Infectivity of the fluorescent P.plecoglossicida was much the same as the wild type. The cells of fluorescent P.plecoglossicida attaching to the body surface of ayu were easily detected under a fluorescence microscope. It was revealed that they adhered predominantly to the micros-injuries in the skin and fins.3) Attachment and invasion of Glugea plecoglossi (Microspora) spores and Thelohanellus hovorkai (Myxozoa) actinospores on the body surface of host fishes were investigatea. G.plecoglossi spores labeled with Uvitex 2B were observed to attach on the micro-injuries or rainbow trout stained with trypan blue. In situ hybridization (ISH) protocol with DIG-labeled oligonucleotide probes designed from SSUrRNA of G.plecoglossi was established to detect all stages of G.plecoglossi. ISH revealed that G.plecoglossi spores discharged the polar tube in the subcutaneous tissue and injected the infective sporoplasm. T.hovorkai actinospores labelled with CFSE were observed to invade not in the skin (regardless of the presence of micro-injuries) but in the gills or carp. These results showed that the relationship between infections with fish parasites and micro-injuries on the host fishes depended on the parasite species. Less

1)采用实时荧光定量聚合酶链反应（Q-PCR）技术，研究了杀褶舌假单胞菌（Pseudomonasplecoglossicida）感染香鱼组织的分布。将鱼浸入10^7CFU/mL的杀多舌类囊原虫悬浮液中15 min。皮肤、鳃、肝脏、脾脏、肾脏和血液经Q-PCR检测，皮肤和/或鳃可能是细菌的入口，因为在感染后1和3 h发现了杀褶舌类假单胞菌，分别然而，在感染后6 h，细菌更多地集中在肝、脾和肾中。这些结果表明，刺舌拟单胞菌在感染后6 h内即可成功入侵香鱼。感染后48小时发生败血症，2）将绿色荧光蛋白（GFP）基因插入穿梭质粒pME 4150中，构建GFP载体质粒。不 ...更多信息在非选择性条件下，pSKTO 3基因在杀多舌类青霉中的表达量最高，且稳定性较好。荧光P.plecoglossicida的生物活性与野生型几乎相同。在荧光显微镜下，可以很容易地观察到贴附在香鱼体表的荧光杀舌类棘球绦虫细胞。结果表明，它们主要附着在皮肤和鳍的微小损伤处。3）研究了小孢子虫Glugea plecoglossi（Microspora）孢子和粘虫Thelohanellus hovorkai（Myxozoa）放线孢子在宿主鱼体表的附着和侵入情况。观察到用Uvitex 2B标记的多舌克氏菌孢子附着在微损伤或用台盼蓝染色的虹鳟鱼上。用地高辛标记的寡核苷酸探针设计的SSUrRNA的plecoglossi的原位杂交（ISH）协议，建立了检测所有阶段的G. plecoglossi。原位杂交结果显示，多舌格孢菌孢子在皮下组织中排出极管，注入感染性孢子质。观察到CFSE标记的T.hovorkai放线孢子不侵入皮肤（无论是否存在微损伤），而是侵入鳃或鲤鱼。这些结果表明，寄生虫感染与宿主鱼的微损伤之间的关系取决于寄生虫种类。少