Carp (cyprinus carpio) hematopoietic cells collected from the kidney-hematopoietic tissue were cultured. After 3-5 days, colonies consisting of a few to 30 small round cells were observed. The number of cells in the colonies increased steadily. Colonies having several hundreds of cells were observed later. Colonnies were formed on an adherent cell layer, which is possibly acting as feeder cells for the colony. Most cells of the adherent layer were 0.1-1.0 mm in diameter and contained 3-200 oval or round nuclei, similar in appearance to that of multinucleated giant cells.
The proliferated cells in the colonies were separated from the colony into the culture medium. These floating cells were constantly produced for more than 1 month while half of the medium was renewed at 1-5 day intervals. Morphologically, the majority of the floating cells had features of immature type of cells which were mainly round cells with basophilic cytoplasm and a round nucleus. Mitotic figures were frequently observed.

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This study was undertaken to investigate the immune response of eel Anguilla japonica against formalin-killed cells (FKC) of Cytophaga columnaris. Eels were vaccinated by immersion or injection with FKC. The immune response of the eels was studied 2 weeks after the vaccination. Prior to the challenge, the eel skin was damaged by wiping with alcohol absorbed cotton. For the challenge, the fish were immersed in a suspension of C. columnaris at a density of 1.0 × 10⁷ CFU/ml for 15 min.
The survival rates of the immersion, injection and control groups were 60, 20 and 0% respectively. In the immersion group skin lesions appeared later than in the other groups, and infiltration of leucocytes in skin lesions occurred at 12 h after the challenge, while no such leucocyte infiltration occurred in other groups. Inhibition activity of bacterial adhesion in the skin was demonstrated in the immersion fish. However, no agglutinating antibody was detectable in the serum or mucus of fish vaccinated by immersion. These results indicate that immersion immunization can activate the eel skin defense system.

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Histopathological comparisons were made between ayu (Plecoglossus altivelis) and carp (Cyprinus carpio) artificially infected with the causative fungus of mycotic granulomatosis, Aphanomyces piscicida NJM 8997. Ayu showed typical pathology of mycotic granulomatosis. On the other hand, in carp, no gross signs of inflammatory responses were observed during the experimental period of 20 days; however, mycotic granulomatous lesions were observed histologically in the inoculated site of the trunk muscles. It was considered histopathologically that carp responded to the inoculated fungus more quickly and intensively than ayu. In addition, the morphometrical features of fungal hyphae in the lesions of carp also suggested that fungal activities were suppressed by the inflammatory responses. Therefore, it was concluded that lesions in the trunk muscles of carp were restricted to a smaller area by their defense mechanisms, while the hyphae penetrated to the neighboring tissues in ayu because of weaker defense mechanisms.

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Infectivity of baculoviral mid-gut gland necrosis virus (BMNV) to larvae of 5 crustacean species was examined by water-borne inoculation.
Penaeus monodon, P. chinensis and P. semisulcatus were infected with BMNV, but Metapenaeus ensis and Portunus trituberculatus showed no evidence of the infection. P. monodon were demonstrated to be highly susceptible to the virus, developing many hypertrophied nuclei of the mid-gut gland epithelial cells. P. chinensis and P. semisulcatus had lower susceptibility to the virus, showing no growth retardation and no significant mortality, and the infection was considered to be transient.

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A total of 27 clones of hybridoma cells secreting monoclonal antibodies (MAbs) against VDV, the causative agent of viral deformity in yellowtail Seriola quinqueradiata, were generated. These MAbs reacted to VDV-infected CHSE-214 cells but not to uninfected CHSE-214 cells by immunofluorescence test. By using these MAbs, 2 classes of polypeptides (VP2, VP3) were characterized by radio-immunoprecipitation test or Western blot. Thirteen MAbs among 24 MAbs reactive to VP2 polypeptide neutralized virus infectivity but the other 3 MAbs specific to VP3 did not neutralize the infectivity. The reaction patterns of the MAbs obtained to the 3 reference strains (VR-299, Sp and Ab) of IPNV were variable. However, neutralization epitopes were conserved on VP2 of both VDV and IPNV. Six isolates of birnaviruses from marine cultured fishes were studied for reactivities of the MAbs by immunofluorescence test. The MAbs showed same reaction patterns to an isolate (YAV-1) from yellowtail and an isolate (H-1) from Japanese flounder Paralichthys olivaceus as those of VDV by immunofluorescence test. However, some MAbs did not react to the other 4 birnavirus isolates from Japanese flounder.

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We have developed a method for detection of penaeid rod-shaped DNA virus (PRDV, formerly RV-PJ), the causative agent of penaeid acute viremia (PAV) of kuruma shrimp, by polymerase chain reaction (PCR). Two pairs of PCR primers (P1/P2 and P3/P4) were prepared, based on the nucleotide sequence of the PRDV genome. PCR using either P1/P2 or P3/P4 specifically amplified PRDV DNA. When PCR products amplified with P1/P2 were subjected to nested PCR with P3/P4, a higher sensitivity was seen than with PCR using P1/P2 or P3/P4 alone. The nested PCR was capable of detecting PRDV from a symptomatic kuruma shrimp that had been experimentally infected with low doses of PRDV, excretions from experimentally infected kuruma shrimp, and other crustaceans in a culture pond where spontaneous PAV occurred.

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The fluorochrome Uvitex 2B [4, 4-BIS (2-di (2-hyroxyethyl) -amino-4- (3-sulfophenylamino) -1, 3, 5-triazine-6-ylamino) -stilbene-2, 2-disulfonic acid, sodium salt] stain was used for detection of Microsporidium seriolae (Protozoa : Microspora), the causative agent of beko disease of yellowtail Seriola quinqueradiata and goldstriped amberjack S. lalandi juveniles. For detection of the parasite, microscopical fluorescence examination for spores in the Uvitex 2B-stained smears of trunk muscle homogenates was much more sensitive than the conventional visual inspection for “cysts” in the trunk muscle. The Uvitex 2B-H & E stain of deparaffinized sections was applicable to the examination for the sporulation sequence in “cysts” and spore dispersal into adjacent tissues or other organs. Thus, Uvitex 2B stain was found to be useful not only for a rapid and sensitive diagnosis of beko disease but also for histopathological studies of microsporidian infections.

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