A microplate enzyme immunoassay (MEIA) was developed for the rapid, sensitive and specific detection of monoclonal antibody-producing hybridomas against IHNV at the first screening. Ninety-six-well microplate cultures of chinook salmon embryo (CHSE-214) cells were infected with IHNV and incubated at 15°C to permit antigen development. The hybridoma supernatants were added to the plates containing 95% ethanol-fixed IHNV-infected CHSE-214 cells as the first antibodies. Peroxidaseconjugated antimouse Ig goat serum was applied as the second antibody and visualization was performed using diamino-benzidine. Under microscopy, positive antigen-antibody reaction was apparently observed by a brownish staining of IHNV-infected CHSE-214 cells. Thus, this method was found highly efficient and sensitive for the screening of monoclonal antibody.
Histopathological studies were made on a virus-infected coho salmon (Oncorhynchus kisutch) characterized by an abnormal swimming. Both naturally and artificially infected coho salmon showed necrosis in the kidney, including the hematopoietic elements and renal tubular epithelial cells, and also degenerative changes of melanomacrophage centers. In the brain, vacuolar degeneration with necrosis of nerve cells, congestion of blood vessels and degenerated nerve fibers were observed. By the EM, viral particles were seen in the cytoplasm of tubular cells and the macrophages in the kidney and in the axons of necrosed nerve cells in the brain.
To clarify the production kinetics of antigenicity of virion polypeptides of yellowtail ascites virus (YAV), time course of the production of capsid proteins, VP2 and VP3, in infected CHSE-214 cells was surveyed by western blotting using polyclonal antisera against purified virion, VP2 or VP3. Pre-VP2 and VP3 were detected at 3h after infection, whereas matured VP2 appeared after 7h. The virus titer in the culture supernatant increased at 10h after infection. These results suggest that the cleavage of polyprotein to pre-VP2 and VP3 occurs in early period of infection and the VP2 maturation occurs after eclipse stage.In serological analysis, anti-VP3 serum did not neutralized the virus. Reactivity of anti-VP3 serum in ELISA increased when virion was treated with SDS.
Pathogenicity of an isolate (CSH9003) of salmonid herpesvirus 2 from maricultured coho salmon (Oncorhynchus kisutch) to coho salmon, masu salmon (Oncorhynchus masou) and rainbow trout (Oncorhynchus mykiss) was compared with those of NeVTA, OMV and YTV. Injections of CSH9003 resulted in high mortality in coho salmon and lower mortality in masu salmon, while no mortality was observed in rainbow trout. In contrast, NeVTA from kokanee and OMV and YTV from masu salmon showed high virulence to masu salmon and lower virulence to coho salmon and rainbow trout. The susceptivility of coho salmon to artificial infection with CSH9003 decreased with the size of fish, although CSH9003 showed lethal pathogenicity to even fish weighing 200g. Mortality caused by the injections of CSH9003 and CSH9104 both isolated from dead coho salmon were similar to that caused by CSH9117-T isolated from the basal cell tumor tissue. In survivors of CSH9003-injected coho salmon fry, the basal cell tumor was found on the gill filament about 18 months after inoculation.
Yellowtail (Seriola quinqueradiata) weighing 40-92g were experimentally infected with an oxolinic acid (OA) resistant strain of Pasteurella piscicida, and then the discharge of the bacterial cells from the fish was determined by using trypticase-soy agar containing OA. The infected fish began to shed P.piscicida cells one or two days before death. The number of cells shed from dead fish reached 107-109CFU/fish·10min, and the same level of bacterial shedding was kept for about 5 days after death. It was also confirmed that the released bacteria (2.4×103CFU/ml) had high infectivity to yellowtail.
Under laboratory conditions at 25°C, the monogenean Neobenedenia girellae (Hargis, 1955) rapidly developed on Japanese flounder (Paralichthys olivaceus), and it took 15-17 days from egg to maturation.The whole process of egg formation from the release of oocytes from the germiduct to laying of egg capsule required a mean of 73sec. At room temperatures between 27-30°C, large adults laid a mean of 35.4eggs/h, while smaller ones a mean of 12.2eggs/h. Eggs began to hatch after incubation for 5-6 days at 25°C with natural cycle of illumination, but no hatching occurred at 15°C. It took 10-11 days to reach sexual maturity where the minimum body size for an egg-laying adult was 2.1mm. When compared with other benedeniine species, N.girellae required a shorter time and a smaller body size to attain maturity and showed a high level of egg output. One-day old larvae were found only on the fins of Japanese flounder.As the parasites grew, they were found not only on the fins but most commonly on the dorsal and ventral body surface of fish, suggesting migration of the parasite from the fins.
The immune response of Japanese flounder (Paralichthys olivaceus) against Neobenedenia girellae (Hargis, 1955) (Monogenea : Capsalidae) was investigated. An acquired protection of fish against secondary infection with the parasite was demonstrated by a reduction in the number and body size of parasites on primed fish which had previously been infected with N. girellae and treated by freshwater bath. In the same experiment, however, no raise in the level of antibody in the sera of primed fish was detected by the enzyme-linked immunosorbent assay (ELISA). In another experiment, although antibody was detected by ELISA in the sera of fish immunized by injection with sonicated parasite antigen, there was no significant difference in the parasite counts between antigen-injected and PBS-injected control fish when challenged with oncomiracidia of N. girellae. These results indicate that the protection induced by the previous infection was not associated with the humoral antibody.