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

1)应用实时荧光定量聚合酶链式反应(Q-PCR)技术研究了舌假单胞菌在浸泡攻击后不同组织中的分布。将鱼浸泡在107CFU/mLPlecoglossicida悬浮液中15min。分别于感染后1、3、6、12、24、48、72h(P.I.)对皮肤、鳃、肝、脾、肾和血液进行了Q-PCR检测。皮肤和/或鳃可能是细菌的入口，因为在P.I.1h和3h分别发现了P.plecoglossicida。6h后，肝、脾、肾组织中细菌含量较高。这些结果表明，黑斑潜蝇能在6h内成功入侵玉米象。(2)将绿色荧光蛋白(GFP)基因插入穿梭载体pME4150中，构建绿色荧光蛋白(GFP)表达载体。T…在非选择性培养条件下，携带pSKT03的P.plecoglossicida的GFP表达水平最高，且稳定性较好。该荧光菌株的感染性与野生型相同。在荧光显微镜下可以很容易地观察到附着在体表上的荧光巴氏并殖吸虫细胞。3)研究了小孢子虫孢子和粘虫放线孢子在寄主鱼体表面的附着和入侵情况。观察到Uvitex 2B标记的长舌鱼孢子附着在微损伤处或台盼蓝染色的虹鲑鱼上。建立了地高辛标记的寡核苷酸探针原位杂交(ISH)技术，用于检测棉铃虫的各个发育阶段。ISH结果显示，扁穗革兰氏菌的孢子排出皮下组织中的极管，并注入侵染性孢子浆。用CFSE标记的Hovorkai放线孢子被观察到侵入的不是皮肤(不管是否存在微损伤)，而是鳃或鲤鱼。这些结果表明，鱼类寄生虫感染与寄主鱼类微损伤的关系取决于寄生虫的种类。较少

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