Tunic morphology and viral surveillance in diseased Korean ascidians: Soft tunic syndrome in the edible ascidian, Halocynthia roretzi (Drasche), in aquaculture.

'Soft tunic syndrome' causes mass mortality in the edible ascidian Halocynthia roretzi in Korean and Japanese aquaculture. In histopathological comparison, there were no specific differences between diseased specimens from Korea and Japan, indicating that soft tunic syndrome occurring in Korea and Japan is the same disease. No bacterial or protozoan cells were microscopically detected in either healthy or diseased tunics suggesting they are not the direct causes of soft tunic syndrome. Attempts were made to isolate virus from affected ascidians taking into account temperature conditions in which soft tunic syndrome is most prevalent in the field. However, no viruses were isolated from diseased or non-diseased specimens using chinook salmon embryo (CHSE-214), flounder fin (FFN) or epithelioma papillosum cyprini (EPC) cell lines.

Antigenic differences of the scuticociliate Miamiensis avidus from Japan.

In Japan and Korea, outbreaks of scuticociliatosis have frequently occurred in Japanese flounder, Paralichthys olivaceus. Morphological observations and small subunit rRNA gene sequences have shown that the causative agent of scuticociliatosis in the flounder is Miamiensis avidus (syn. Philasterides dicentrarchi). In this study, we elucidated the antigenic differences between six Japanese M. avidus isolates as an initial step toward developing an effective vaccine against the disease. Four Japanese flounder isolates (IyoI, Nakajima, JF05To and Mie0301 isolates), one spotted knifejaw, Oplegnathus punctatus, isolate (SK05Kyo), and one ridged-eye flounder, Pleuronichthys cornutus, isolate (RF05To) were subjected to serological analysis. Antisera against IyoI, SK05Kyo, Nakajima and Mie0301 isolates were raised in rabbits and used for immobilization assays and Western blotting. Immobilization assays showed that the six isolates could be divided into three groups, tentatively designated serotype I for IyoI, JF05To, RF05To, SK05Kyo, serotype II for Nakajima and serotype III for Mie0301. Western blotting results supported these three serotypes, with marked similarities in the banding profiles of IyoI, JF05To, RF05To and SK05Kyo isolates, which were distinct from the Nakajima and Mie0301 isolates. Three isolates, IyoI, Nakajima and Mie0301 that were selected as representatives of each serotype, were highly pathogenic to Japanese flounder by experimental infection. Based on these findings, we propose that there are at least three M. avidus serotypes in Japan.

Lymphocystis disease virus persists in the epidermal tissues of olive flounder, Paralichthys olivaceus (Temminch & Schlegel), at low temperatures.

Olive flounder artificially infected with lymphocystis disease virus (LCDV) were reared at 10, 20 and 30 degrees C for 60 days, to compare LCD-incidence. In the fish reared at 20 degrees C, lymphocystis cells appeared on the skin and fins at 35 days post-challenge, and the cumulative LCD-incidence was 80% at 60 days. High levels of LCDV, with a mean polymerase chain reaction (PCR) titre of 10(6) PCR-U mg(-1) tissue, were detected in the fins and skin of LCD-affected fish at 20 degrees C, but were not detected in the spleen, kidney, brain and intestinal tissues of these fish. No LCD clinical signs were observed in the fish reared at 10 degrees C and 30 degrees C; however, a low level of LCDV (10(3) PCR-U mg(-1) tissue) was detected in the fins and skin of these fish. By increasing the rearing temperature from 10 to 20 degrees C, lymphocystis clusters appeared on the skin and fins of the fish with no previous LCD clinical signs within 33 days after the temperature change. It was shown that permissive cells for LCDV infection exist in the epidermis of olive flounder. At low temperatures, small amounts of LCDV were able to persist over a period extended for a further 45 days in the fish epidermis, even though the fish showed no LCD clinical signs. The optimum growth temperature of LCDV is near 20 degrees C.

Proliferation of kuchijirosho causative agent in a fugu-derived cell line.

Kuchijirosho is a fatal disease of commercially cultured fugu, Takifugu rubripes. The transmissible nature of kuchijirosho strongly suggests that an infectious pathogen is the causative agent. Because it is filtrable, the agent is thought to be a virus; however, it has not yet been identified. The lack of a permissive cell line for the putative kuchijirosho-causing agent (KCA) has hindered research on the identification of this pathogen. We inoculated brain extract prepared from kuchijirosho-affected fugu onto an established fugu cell line, fugu eye, and observed that cytopathic effect appeared 7 days after inoculation. Injection of the culture medium of infected fugu eye cells into fugu resulted in the onset of kuchijirosho, indicating that fugu eye cells are able to proliferate KCA. An infectious fraction separated by sodium iottalamate density gradient centrifugation showed a density of 1.15 g mL(-1) equivalent to that of KCA derived from affected fugu brain. To determine whether the genome of KCA is RNA or DNA based, nucleotide synthesis inhibitors were applied to inoculated fugu eye cell line to influence the production of KCA. 5-Fluorouracil but not IUdR showed a concentration-dependent inhibition of KCA yield. These results suggest that KCA is an RNA virus.

Susceptibility of marine fish species to a megalocytivirus, turbot iridovirus, isolated from turbot, Psetta maximus (L.).

Turbot iridovirus (TBIV), a member of the genus Megalocytivirus in the family Iridoviridae, was isolated from diseased turbot, Psetta maximus (L.), in Korea in 2003. In this study, experimental infection of turbot, Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel), and rock bream, Oplegnathus fasciatus (Temminck & Schlegel), with TBIV was performed to evaluate the viral susceptibility of these fish species. After virus exposure, the mortalities of turbot reared at 22 and 25 degrees C were 60% and 100%, respectively, suggesting that TBIV is the causative agent of the mass mortality of turbot that occurred in Korea in 2003. Moreover, TBIV was detected in Japanese flounder and rock bream by polymerase chain reaction after experimental infection (26 days post-inoculation) despite no viral pathogenicity in these fish, suggesting that these two fish species are also susceptible to the virus. It is possible that horizontal transmission of TBIV occurs among these three fish species because turbot is routinely cultured with Japanese flounder and rock bream in Korea.

A new genotype of lymphocystivirus, LCDV-RF, from lymphocystis diseased rockfish.

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease. In this study, nucleotide sequences of the major capsid protein (MCP) gene were analyzed among LCDV isolates from Japanese flounder and rockfish. A phylogenetic tree revealed three clusters for lymphocystiviruses. The first cluster included Japanese flounder isolates; the second cluster consisted of rockfish isolates; and the remaining one consisted of LCDV-1. Nucleotide sequence identities were > or =99.6% among Japanese flounder isolates and 100% among rockfish isolates, while between each cluster they were < or =85.2%. Experimental infections with Japanese flounder and rockfish isolates revealed that Japanese flounder and rockfish were infected by the respective homologous isolate but not by the heterologous isolate. These findings suggest that at least three genotypes exist in the genus Lymphocystivirus.

Experimental infection of several fish species with the causative agent of Kuchijirosho (snout ulcer disease) derived from the tiger puffer Takifugu rubripes.

Kuchijirosho (snout ulcer disease) is a fatal epidemic disease which affects the tiger puffer, Takifugu rubripes, a commercial fish species in Japan and Korea. To assess the possibility that non-tiger puffer fish can serve as reservoirs of infection, 5 fish species were challenged by infection with the extracts of Kuchijirosho-affected brains from cultured tiger puffer: grass puffer T. niphobles, fine-patterned puffer T. poecilonotus, panther puffer T. pardalis, red sea bream Pagrus major, and black rockfish Sebastes schlegeli. When slightly irritated, all these species, especially the puffer fish, exhibited typical signs of Kuchijirosho, i.e., erratic swimming, biting together and bellying out (swelling of belly), as generally observed in tiger puffers affected by Kuchijirosho. Although the mortalities of the 2 non-puffer species were lower, injection of the extracts prepared from the brains of both inoculated fish into tiger puffer resulted in death, indicating that the inoculated fish used in this experiment have the potential to be infected with the Kuchijirosho agent. Condensations of nuclei or chromatin in the large nerve cells, which is a major characteristic of Kuchijirosho, were histopathologically observed to some extent in the brains of all kinds of puffer fish species infected. These findings suggest that the virus can spread horizontally among wild and cultured puffers and even among fishes belonging to different orders.

Genomic organization, expression, and analysis of the troponin C gene pat-10 of Caenorhabditis elegans.

We have cloned and characterized the troponin C gene, pat-10 of the nematode Caenorhabditis elegans. At the amino acid level nematode troponin C is most similar to troponin C of Drosophila (45% identity) and cardiac troponin C of vertebrates. Expression studies demonstrate that this troponin is expressed in body wall muscle throughout the life of the animal. Later, vulval muscles and anal muscles also express this troponin C isoform. The structural gene for this troponin is pat-10 and mutations in this gene lead to animals that arrest as twofold paralyzed embryos late in development. We have sequenced two of the mutations in pat-10 and both had identical two mutations in the gene; one changes D64 to N and the other changes W153 to a termination site. The missense alteration affects a calcium-binding site and eliminates calcium binding, whereas the second mutation eliminates binding to troponin I. These combined biochemical and in vivo studies of mutant animals demonstrate that this troponin is essential for proper muscle function during development.

Platelet-dependent thrombin generation in patients with diabetes mellitus: effects of glycemic control on coagulability in diabetes.

OBJECTIVES: This study sought to assess the usefulness of platelet-dependent thrombin generation as an index of coagulability in diabetes and to determine the effect of glycemic control on coagulability in diabetes. BACKGROUND: It is important to investigate the interaction of platelets and the coagulation factors to clarify the processes of the coagulation system in detail. METHODS: Platelet-rich plasma (150 X 10(9)/liter), 0.5 ml, was prepared, and 40 mmol/liter of calcium chloride was added to initiate clotting. S-2238 was added to each sample in a microtiter plate every 10 min, and the absorbance of the released color product at 2 min was measured spectrophotometrically at a wavelength of 405 nm using a microtiter plate reader as thrombin generation. We measured the platelet-independent thrombin generation in patients with non-insulin-dependent diabetes mellitus grouped according to glycemic control. RESULTS: Platelet-dependent thrombin generation at 30 min after calcium chloride addition was significantly higher in 23 patients with poorly glycemic-controlled non-insulin-dependent diabetes mellitus without complications, such as diabetic retinopathy, nephropathy and neuropathy (hemoglobin [Hb] A1c >/= 9.0%) than in 46 healthy normal subjects (448 +/- 75 vs. 165 +/- 28 mU/min, p < 0.001). Thrombin generation in 31 well controlled diabetic patients without complications (Hb A1c < 9.0%) was intermediate (240 +/- 72 mU/min) between those of the poorly controlled group and healthy normal subjects. Platelet-poor plasma from diabetic patients increased platelet-dependent thrombin generation in normal subjects. CONCLUSIONS: Coagulability is evidently enhanced in patients with non-insulin-dependent diabetes mellitus compared with that in healthy normal subjects on the basis of assessments of the platelet-dependent thrombin generation, and good glycemic control may help to correct a hypercoagulable state in diabetic patients.