Attempts were made to cultivate Cryptocaryon irritans in vitro at 23-25°C. Attachment of theronts and subsequent enlargement into trophonts were achieved in two experiments using strips of trypticase soy agar (TSA, supplemented with 3% NaCl) as an attachment substrate in filtered seawater. In the third experiment, transformation of theronts into trophonts was achieved in an enriched liquid medium composed of 50% filtered seawater, 30% Leibovitz L-15 and 20% fetal calf serum without attachment onto the TSA. Sizes (mean ±SD) of the trophonts, 114.6 ± 57.9 μm to 295.9 ± 130 μm, were from a recorded size range (50 to 700μm) of the parasite in vivo. Although only limited numbers of theronts (0.28-1.71%) transformed into trophonts, these results showed that the in vitro culture of C. irritans is potentially feasible as evidenced by the enlargement of the trophonts within the in vivo size range using either a solid medium as an attachment substrate or a liquid medium without attachment. There is a need, however, to determine essential factors that influence the transformation of the trophonts into viable tomonts capable of producing theronts.
This study was made to clarify a possibility of vertical transmission of bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis. The presence of Flavobacterium psychrophilum on the surface and in the contents of unfertilized and fertilized eggs, which were obtained from ayu naturally carrying F. psychrophilum, was examined by a culture method. The bacterium was detected on the egg surface of 19 out of 65 unfertilized egg samples and of 5 out of 30 fertilized egg samples (300 eggs/sample). On the other hand, F. psychrophilum was not detected from the surface of eggs disinfected by povidone-iodine (5 ppm, 10 min) or hydrogen peroxide (150 ppm, 30 min). The bacterium was not detected in the contents of these surface-disinfected eggs. These results suggest that F. psychrophilum is not transmitted through intra-ovum infection in ayu eggs. Therefore, disinfection of eggs is useful to prevent the vertical transmission of BCWD in ayu hatcheries.
Two cDNA libraries were prepared from Japanese flounder Paralichthys olivaceus kidney cells that had been treated with LPS or a combination of ConA and PMA. From these two libraries, we sequenced 373 and 379 clones, respectively. Of these 752 clones, 109 clones were characterized as immune-related. Among the immune-related cDNAs, the following have not been previously reported in fish : NK-lysin, perforin, complement C1 q, CD9 antigen, CD63 antigen, CD82 antigen, ISGF-3, IP-30, G-CSF, equistatin precursor, TLS-CHOP, fas ligand and CARD4. This study highlights the success of using ConA/PMA and LPS as mitogens to increase the number of immune-related genes expressed in Japanese flounder and demonstrates that different stimuli induce the expression of different immune-related genes.
We investigated the infection dynamics of adult Neoheterobothrium hirame in young Japanese flounder Paralichthys olivaceus (20-50 cm in total length) in the Joban Sea, eastern Japan. The prevalence and abundance of the parasite, which were statistically irrelevant to host factors namely both wild/released and male/female, showed seasonal fluctuations and peaked in winter. The oldest record of the parasite in the Joban Sea was found in 1997. Although the infection level was high from 1998 to 2002, it decreased dramatically in 2003. The factors influencing the adult parasite infection level were unclear, but one of the possible factors would be the continuous low water temperature from August 2002 to February 2004, especially in the summer of 2003, when new infections should have occurred. The low temperature seems to have decreased the egg-laying rate of N. hirame and consequently lowered its infection level.
Mass mortalities caused by akoya oyster disease have been occurring in cultured Japanese pearl oyster Pinctada fucata martensii in western Japan. Although the involvement of some causative agents was suggested, the cause of the disease is still unidentified. In this study, an experimental infection was performed by cohabitation of the diseased and healthy pearl oysters. A high mortality and characteristic disease conditions were observed in the test group. This result reconfirmed that this disease is caused by a certain infectious agent. To clarify the tissue distribution of the causative agent in diseased pearl oysters, two experimental infections were performed by transplantation of pieces of various tissues or injection of homogenates of various tissues from diseased pearl oysters into healthy pearl oysters. The hemolymph, mantle and adductor muscle showed high infectious titers compared with those of the heart, digestive gland and hemocytes. The mantle had the highest infectious titer. Moreover, injection of the supernatant of a mantle homogenate also transmitted the disease to test pearl oysters. These results suggest that the causative agent is concentrated in the mantle.
We developed a polymerase chain reaction (PCR) method to detect flounder herpesvirus (FHV), the causative agent of viral epidermal hyperplasia of larval Japanese flounder Paralichthys olivaceus. The developed PCR method was specific to FHV and no PCR products were obtained in other fish-pathogenic herpesviruses and flounder-pathogenic viruses tested. FHV was detected by the PCR test with a sample containing 100 or more epidermal cells, which were derived from flounder larvae with epidermal hyperplasia. Four samples of diseased flounder larvae, which were collected from natural mass mortalities and diagnosed as viral epidermal hyperplasia by conventional methods, were positive by the PCR test for FHV. These results show that the present PCR method is useful for rapid and reliable diagnosis of fish clinically or subclinically infected with FHV.
To purify the kuchijirosho causative agent derived from brains of kuchijirosho-affected tiger puffer Takifugu rubripes, the method of density gradient centrifugation using iodinated contrast medium, Angio-Conray (solium iotalamata solution), was used. The extracts of kuchijirosho-affected brains were loaded on 3-55% gradient of Angio-Conray, and centrifuged at 280, 000 ×g for 2 h. The lethal activity to grass puffer Takifugu niphobles was found in a fraction with a density around 1.096 g/cm3. The infectivity of the extracts of kuchijirosho-affected brains was lost by treatment with ether, UV-irradiation, β-propiolacton or proteinase K.
Tiger puffer experimentally infected with Heterobothrium okamotoi was given gradual changes of water temperature, and the effect of temperature on the production and viability of the parasite eggs was examined. Among 10, 15, 20, 25 and 30°C, the highest egg production rate was observed at 25°C. While most of eggs produced at 10 or 20°C hatched at 20°C- incubation, those produced at 30°C decreased in hatching rate at 20°C- or 30°C- incubation. Eggs produced at 26°C or more contained morphologically abnormal ones. These results indicate that the optimal temperature for the egg production of H. okamotoi is approximately 25°C.