Infectious pancreatic necrosis virus (IPNV) was infected in CHSE-214 cells, and viral antigens in the cells stained with indirect immunofluorescence method were monitored by flow cytometer during early multiplication stage of the virus. Viral antigen could first be detected at 2 h post infection (p.i.), and the larger numbers of labeled CHSE-214 cells were obtained at 8 and 24 h p.i. in both VR-299 and Sp strains of IPNV examined. The detection method is fast and sensitive and yields quantitative results on single cell basis.
Hematological and histological studies were carried out on yellowtail, Seriola quinqueradiata, experimentally infected by intravenous injection of the bacterium causing jaundice of cultured yellowtail. Hematocrit values and blood hemoglobin concentrations of the experimentally infected fish steadily decreased during 5 days after inoculation. In contrast, plasma hemoglobin and total bilirubin concentrations were maintained at a normal level during 3 days, but rapidly increased on the fifth day after inoculation. Histopathologically, the liver showed moderate focal necrosis and destruction of the endothelium of veins in moribund fish. The most extensive changes were observed in the spleen and kidney : severe anemia and necrosis in the splenic hematopoietic tissue, and degeneration and necrosis in the renal tubules and hematopoietic tissue were found. These pathological changes were similar to those observed in naturally infected fish. By indirect fluorescent antibody technique the bacteria causing jaundice were frequently detected in the spleen and kidney, but not in the hepatic parenchyma. These findings suggest that in jaundiced yellowtail destruction of red blood cells by the causative bacteria resulted in increases in serum hemoglobin and bilirubin concentrations.
An antiviral substance of a high molecular weight, low cytotoxity and potent virucidal activity was purified from the culture supernatant of a marine Alteromonas sp. 48HS-27. Maximum production of this antiviral substance by the strain in MCYG broth was attained by 72 h-incubation at. 25°C. By the purification procedure involving ultrafiltration, precipitation with ammonium sulfate and acetone, gel filtration and native-polyacrylamide gel electrophoresis (PAGE), a polypeptide (48HS-27A) with antiviral activity was obtained at a 270-fold purification with 6.20% yield from the culture supernatant. Molecular weight of the purified 48HS-27A was estimated as approximately 52 kDa by both native and sodium dodecyl sulfate (SDS) PAGE. The 50% infection inhibitory concentrations of this substance were from 0.09 to 2.51μm/ml against one herpesvirus and five rhabdoviruses, whereas the minimal cytotoxic concentration of the substance was 144μm/ml against FHM and CHSE-214 cells. The purified 48HS-27A had proteolytic activity against casein and bovine serum albumin.
An undescribed virus was isolated from the brain of coho salmon (Oncorhynchus kisutch), iwana (Salvelinus pluvius), rainbow trout (O. mykiss) and ayu (Plecoglossus altivelis), and ovarian fluid of masu salmon (O. masou) cultured in the northern part of Japan. The virus was isolated from both juveniles and adult fishes. Diseased fish showed abnormal swimming movement and were lethargic. The virus replicated, inducing cytopathic effects and lysis in susceptible cell lines at temperatures of 5 to 25°C. Persistent infection was observed in several fish cell lines. The virus particles were enveloped, and were icosahedral in shape with about 80 nm in diameter with a central electron-dense core. The virus was replicated with IUdR and BVdU, and inclusion bodies in the infected cells were stained with acridine orange. The virus density was 1.155g/ml and the viral RNA was 7.3kb in length. The virus was stable to pH, diethyl ether and chloroform. The virus was not neutralized by antisera against known 6 fish viruses.
Pathogenicity of the new virus isolated from the brain of coho salmon Oncorhynchus kisutch was investigated by waterborne and intramuscular inoculation to coho salmon, masu salmon O . masou, iwana Salvelinus pluvius, steelhead trout O. mykiss, and ito Hucho perryi. Typical disease signs of spinning swimming and lethargic behavior were observed in the experimentally infected fishes. The cumulative mortality of coho salmon fry for 60 days bathed with the virus reached 6 to 34%. In intramuscular injection of the virus, cumulative mortality of coho salmon fry reached 51 to 63%. The virus also showed pathogenicity to masu salmon, steelhead trout and iwana. Virus antigen was detected by indirect fluorescent antibody test (IFAT) in the kidney, brain and blood cells of the infected fish.
Kuruma prawns (Penaeus japonicus) were experimentally infected with Vibrio sp. PJ by the oral administration. At 3, 6, 8, 10, 12, 24, 36, and 48 h post-inoculation, prawns were sampled to determine the cell number and distribution of the pathogen in various organs by viable cell count and the enzyme-labeled antibody technique (ELAT). Vibrio sp. PJ was detected by culture method from the stomach and hemolymph at 3 h and from the hemolymph and almost all the organs sampled at 6 h post-inoculation. However, the pathogen started to disappear from all the organs from 8 h to 12 h. At 12 h, it reappeared in the hepatopancreas and lymphoid organs. Twenty-four hours after inoculation, the pathogen was detected from the hemolymph and all the organs except the stomach and gills, and at 36 h the pathogen was found distributed in all the organs. Principally the same distribution pattern of the pathogen was found by ELAT. These results seem to indicate that the process in the pathogenesis of Vibrio sp. PJ infection in orally challenged kuruma prawns consist of five stages namely, establishment of the pathogen, distribution of the pathogen, clearance of the pathogen by host prawn, secondary multiplication of the pathogen, and systemic infection. The results also suggest that the pathogen multiplied in the stomach in the establishment stage and in the hepatopancreas and lymphoid organs in the secondary multiplication stage.
A total of 20 hybridoma clones secreting monoclonal antibodies (MAbs) were established between mouse myeloma cells and spleen cells from mice immunized with red sea bream iridovirus (RSIV) isolated from diseased red sea bream Pagrus major. These MAbs reacted with RSIV-infected BF-2 cells but not with uninfected BF-2 cells in immunofluorescence assays. With these MAbs, 2 classes of RSIV specific polypeptides (230/180 kDa, 20/16 kDa) were characterized by immunoprecipitation and SDS-PAGE. All 20 MAbs reacted with BF-2 cells infected not only with RSIV isolates from various geographic areas, but also with sea bass iridovirus (SBIV) isolated from diseased sea bass Lateolabrax sp. Twelve of the 20 MAbs reacted positively with BF-2 cells infected with Japanese parrot fish iridovirus (JPIV) from diseased Japanese parrot fish Oplegnathus fasciatus. None of the MAbs reacted with EK-1 cells infected with icosahedral cytoplasmic deoxyribovirus (ICD virus) isolated from diseased Japanese eel Anguilla japonica, or with FHM cells infected with Frog virus 3 (FV3) belonging to the family Iridoviridae.
Histopathological changes assoicated with the infection of viral deformity virus (VDV) were described in yellowtail fingerlings, Seriola quinqueradiata, from a hatchery in Nagasaki Prefecture. Advanced congestion in the liver, edema and anemia in the kidney, anemia in the spleen and congestion in the various parts of the brain were observed in naturally diseased fish. Experimentally infected fish showed congestion and focal necrosis in the liver, vacuolar degeneration in tubular epithelial cells and edematous changes in the hematopoietic tissue of kidney, and extensive anemia and necrosis of the splenic tissues. In the brain of fish showing abnormal swimming behavior and deformity, advanced congestion and hemorrhage were found in all fish examined. Histopathological features in experimentally infected fish closely resembled those in naturally diseased fish. These findings suggest that lesions in the brain are related to abnormal swimming behavior and deformity of the diseased fish.
Indirect fluorescent antibody technique (IFAT) usinganti-NeVTA nucleocapsid rabbit serum was applied to rapid diagnosis of salmonid herpesvirus 2 (SH-2) infection in maricultured coho salmon, Oncorhynchus kisutch. Virus-specific fluorescence was seen in nuclei of the CHSE-214 cells infected with NeVTA and coho salmon herpesvirus isolant 9003 (CSH9003). This antiserum could be successfully used for detection of the virus from the liver, heart, spleen and kidney of diseased fish. With IFAT using frozen sections and smears of diseased coho salmon liver, the detecting ratio of the virus was over 90% and comparable to the ratio obtained by virus isolation. In the liver, stored at -18°C for 30 days, extreme loss of viral infectivity occurred and no virus was isolated from the liver stored for 200 days. On the other hand, virus-specific fluorescence could be observed without any decrease of intensity in the smeared samples prepared from the liver stored for 200 days. This method was proved to be a simple, rapid and reliable diagnostic method for SH-2 infection.
Aeromonas salmonicida was isolated from diseased yamame (Oncorhynchus masou) at 15, 20, and 25°C. Ten colonies were taken from each of ten isolates and were subcultured at the corresponding temperatures. Colonies incubated at 15 and 20°C showed strong auto-agglutination but 40% of colonies incubated at 25°C didn't clearly show auto-agglutination. It was also observed that strains isolated from diseased iwana (Salvelinus leucomaenis) at 20°C and subcultured at 25°C showed weak or no agglutination. These observations strongly suggested that A. salmonicida should be isolated and subcultured at 15°C or 20°C in order to retain their auto-agglutinability.
Triploid rainbow trout was compared with diploid one for the susceptibility to IHN, furunculosis and vibriosis, and for the effectiveness of vibrio vaccination. No difference was observed between the triploids and the diploids in the mortality or in the mean days to death when challenged with the pathogens of the diseases. Vaccinated triploids showed significantly lower mortality than triploids without vaccination when challenged with Vibrio ordalii, and there was no difference in the mortality between vaccinated diploids and triploids. These results suggest that susceptibility to those infections and effectiveness of vibrio vaccination are principally the same for both diploid and triploid rainbow trout.
In Japan, seed production techniques have been developed for about 80 species of marine fish and shellfish. However, mass mortalities due to infectious and non-infectious diseases have often occurred in larvae and juveniles reared in hatcheries. Among these problems the viral and bacterial diseases are reviewed in this paper. Since around the middle of 1980's some new viral diseases such as viral epidermal hyperplasia (herpesvirus infection) in the Japanese flounder, viral ascites (birnavirus) in yellowtail, viral nervous necrosis (VNN) (nodavirus) in striped jack and some other fishes, and baculoviral mid-gut gland necrosis (BMN) in kuruma prawn have been reported. It was demonstrated that the selection of virus-free spawners based on the diagnosis by polymerase chain reaction (PCR) could serve as a control measure against vertical transmission of the pathogen in striped jack. Vibriosis, pasteurellosis, gliding bacterial infection and other bacterial diseases have occurred in various marine fishes during their juvenile stages. On the other hand, larval fish most frequently develop intestinal infections represented by bacterial enteritis with Vibrio sp. INFL in the Japanese flounder. Live foods contaminated with pathogenic bacteria have been suspected to serve as an important source of these intestinal infections.