|Budget Amount *help
¥10,500,000 (Direct Cost : ¥10,500,000)
Fiscal Year 2000 : ¥2,900,000 (Direct Cost : ¥2,900,000)
Fiscal Year 1999 : ¥7,600,000 (Direct Cost : ¥7,600,000)
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
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