Enzyme electrophoresis for superoxide dismutase (SOD) and catalase divided 144 Edwardsiella tarda strains into two groups; group 1 contained all the strains isolated from diseased fish in Japan and group 2 included 25 strains from non-disease sources and reference strain ATCC15947T. These groups could be similarly distinguished by a simple catalase test; group 1 showed an instantaneous bubbling, whereas in group 2 bubbling was delayed or weak. PCR-RFLP analysis usingHhaI revealed that the two groups differed in their 16S rDNA genotypes. Furthermore, this analysis subdivided group 1 into two genotypes.
Influences of pH and temperature on lysozyme activity in the plasma from Japanese flounder, Paralichthys olivaceus, and Japanese char, Salverinus leucomaenis, were examined using the turbidimetric assay system. Phosphate buffered solution adjusted at pH 6.1 promoted the highest activity of lysozyme in both fishes. The optimal temperature for the bacteriolytic activity of lysozyme in Japanese flounder and Japanese char was 50°C and 30°C, respectively. The lysozyme activities of both species of fish were heat labile at temperatures higher than 48°C for 30min, and were stable at lower than 30°C for 48 h. These results may be useful in establishing a simple assay system for lysozyme activities in order to evaluate bio-defense level in both fish.
Hatchery-produced juvenile yellowtail (Seriola quinqueradiata) were transferred from an indoor tank to sea netpens on June 20, June 30, July 10 and July 31, 1995 (4 groups), and each group was monitored at about 10 day intervals for Microsporidium seriolae, the causative agent of beko disease. Water temperature gradually increased during the study period, ranging from 20.8 to 30.2°C. In the first 3 fish groups, almost all fish were found to be infected. However, prevalence of infection in the last fish group was much lower than in the other groups. Groups which were transferred later attained their maximum prevalence of infection within a shorter period. Furthermore, we investigated the effects of water temperature and fish size on the development of M. seriolae in juvenile yellowtail. Two different fish size classes (average body weight 0.3 g and 22.0 g) were infected with M. seriolae to a similar extent. In the histological examination, the development of M. seriolae and host recovery were accelerated in fish reared at 25°C compared to those kept at 20°C.
The polymerase chain reaction (PCR) was used to amplify DNA of the red sea bream iridovirus (RSIV). Four oligonucleotide primer sets based on the ATPase gene, DNA polymerase gene, and a Pst I-restriction fragment of RSIV genomic DNA were synthesized. PCR products of the expected size were amplified with each primer set after 30 cycles using template DNA which was extracted from the supernatant of tissue-cultured GF cells infected with RSIV. These amplified products were shown to be specific for the genomic DNA of RSIV by Southern blot hybridization. In addition, PCR products were obtained from the DNAs of the spleen and blood of red sea bream, Pagrus major, artificially infected with RSIV. Furthermore, the PCR products were detected from the tissues of cultured marine fish naturally infected with RSIV and from the supernatant of tissue-cultured GF cells infected with RSIV isolated from cultured marine fish. However, PCR products were not obtained at 3 months post-challenge from the spleens of red sea bream infected by intraperitoneal inoculation of RSIV. None of the PCR products were obtained from the supernatant of tissue-cultured FHM cells infected with frog virus 3 or from the lymphocystis tissue of Japanese flounder, Paralichthys olivaceus, infected with fish lymphocystis disease virus.
Eggs of the monogenean Heterobothrium okamotoi were collected immediately after deposited in Petri dishes, and incubated at 15°C, 20°C and 25°C . The average time required for egg hatching was 11.8 days at 15°C, 7.0 days at 20°C and 5.3 days at 25°C. Effects of different salinities (0, 6.7, 13.4, 20.0, 26.7 and 33.4 ppt) on hatching was examined. Hatching was not affected at the salinities of 6.7-33.4 ppt, but oncomiracidia hatched at 6.7 ppt were apparently inactive, probably because of the low salinity. No embryonic development was observed in the eggs in distilled water, and the embryos eventually died. The morphology of oncomiracidium was described. The oncomiracidium of H. okamotoi and those of previously described Diclidophora species, both in the family Diclidophoridae, had many morphological features in common. The H. okamotoi oncomiracidium could survive much longer than other monogenean larvae, with an average life span of 9.1 days, 7.3 days and 4.7 days at 15°C, 20°C and 25°C, respectively, suggesting that it can retain its infectivity much longer than other monogeneans.
Viral nervous necrosis (VNN) of sevenband grouper Epinephelus septemfasciatus at grow-out stages, which was characterized by upside down swimming or floating behavior, has been spreading in Japan. The present study describes pathogenicity of a nodavirus found in diseased sevenband grouper. When young sevenband grouper or juvenile redspotted grouper E. akaara were challenged by an intramuscular injection with the filtered homogenate of infected organs (brain and eye), abnormal swimming behavior and mortality were produced, but some affected fish recovered from the disease after exhibiting the characterized behavior. Necrosis and vacuolation of the brain and retinal tissues, characteristic to VNN, were produced in the dead and abnormally-swimming fish and the viral antigens were detected in the degenerated nerve cells by FAT with an anti-SJNNV (striped jack nervous necrosis virus) serum. The infection experiment with redspotted grouper also indicated that rearing water temperature (16-28°C ) influenced development of the disease : higher mortality and earlier appearance of the disease signs were observed at higher water temperatures.