Tetraonchus infection of masu salmon, Oncorhynchus masou, was monitored monthly in the culture system at Okutama Branch, Tokyo Metropolitan Fisheries Experimental Station over a period of January 1978 to September 1979 from fry to adult of a single host fish stock artificially spawned there. Two species (T. awakurai and T. oncorhynchi) were always found from the host. It is assumed that the oncomiracidial invasion occurred from spring to autumn, when the water temperature was above 10°C. In T. awakurai infection of fry, both the prevalence and intensity of infection sharply increased in spring, and all the fish were infected in June to August. From September to April, the prevalence of infection stayed about 50% with decreasing mean intensity. It is probable that the lowered infection level in autumn and winter is attributed to the elimination of the parasite by the host. The effect of the elimination was different in degrees among the host fish, leading to over-dispersed distribution of the parasite within the host population. A new infection started in April next spring, but the prevalence of infection did not reach 100 %. The course of infection with T. oncorhynchi was much the same as that of the former species, although the infection level was usually much lower. No relationship was evident between the two tetraonchid species. The infection levels of the two parasites differed greatly from year to year. A possible method for parasite control is also discussed.
In this paper some criteria for the plaque assay of eel virus European (EVE) and infectious hematopoietic necrosis virus (IHNV) in loosely confluent RTG-2 cell monolayers were evaluated. 1. Suitable virus adsorption times for both EVE and IHNV were 60 min. 2. The linear relationship between the number of plaques and the concentration of virus was shown in both EVE and IHNV-RTG-2 cell systems. This indicated that a single infectious viral particle of EVE or IHNV is sufficient to infect a RTG-2 cell. 3. The coefficient of variation of the distribution of plaque numbers was 0.06 in the EVE-RTG-2 cell system and 0.054 in the IHNV-RTG-2 cell system. 4. The number of plaques increased as the volume of viral inoculum increased, but the efficiency of adsorp tion decreased with inocula volumes greater than 0.1 ml. 5. Neither the concentration of agar in agar overlay medium (AOM) nor the incubation periods significantly affected the number of plaques (EVE : 0.5<P, 0.5<P; IHNV : 0.25<P, 0.5<P, respectively). However there was a significant effect of both concentration of agar in AOM and incubation period on the diameter of plaques (EVE : P<0.05, P<0.005 ; IHNV : p<0.005, P<0.01, respectively). These results indicated that loosely confluent RTG-2 cell monolayers gave precise quantitative analysis for EVE and IHNV.
In early summer, 1983, an acute disease characterized by ascites occurred among yellowtail fingerlings, Seriola quinqueradiata, cultured in a farm in the Seto Inland Sea, Japan, and accordingly virological investigations were carried out in July. All three inoculants prepared from the viscera of the fish with ascites produced cytopathic effects (CPE) of pyknosis on RTG-2, CHSE-214 and EK-1 cells within 1.5-5 days after inoculation at 20°C. The virus replicated in CHSE-214 and EK-1 cells at temperature between 5-30°C. Electron micrographs revealed pentagonal or hexagonal particles of 62-69 nm. The virus was resistant to pH 3, pH 11, ether and chloroform and was stable at 56°C for 30 minutes. Replication of the virus was not inhibited by 5-Iodo-2'-deoxyuridine. Neutralization tests with antisera proved that this virus resembled to IPNV. Pathogenicity of the virus was studied using yellowtail fingerlings (average body weight 1.1 g). Fish were exposed to the virus by immersion for 1 hour in seawater containing106.6 TCID50 /ml of the virus. The fish infected with the virus showed mortality of 62 % at water temperature of 20°C and 14 % at 25°C, while controls showed mortality of only 4 % at 20°C and 10% at 25°C, respectively. Moribund fish infected with the virus were often accompanied by severe ascites and hemorrhage in the liver. The virus reisolated from all the infected fish died. The results strongly suggest that this virus was the causative agent of the ascites of yellowtail fingerlings. The agent was tentatively named yellowtail ascites virus (YAV).
Myxobolus buri sp. n. is described from the brain of yellowtails, Seriola quinqueradiata T. et S. with serious scoliosis. The sites of infection were the cerebral cavities and the surfaces of the olfactory lobes, optic lobes and medulla oblongata, Spores of the species were differentiated from those of the other members of the genus Myxobolus on the basis of a through recording of their shape, structure and dimensions by means of light and scanning electron micrography.
Disease-free rainbow trout (Salmo gairdneri) in Europe have a natural (6 S) serum protein capable of inactivating a tissue culture-adapted isolate of infectious pancreatic necrosis virus, IPNV strain Sp (M. DORSON et al., C.R. Acad. Sci., 281, 1435-1438, 1975). We examined five distinct stocks of rainbow trout (from virus-free Canadian facilities) and all expressed a similar inhibitor that neutralized IPNV Sp in fathead minnow (FHM) cells at maximum (reciprocal) mean serum titers ranging from 128 to 512 (80 % plaque reduction, 0.1 ml inocula). Tests with different laboratory strains of virus, including Jasper, Buhl, Reno, Powder Mill, VR 299, OV, and Tellina virus (TV), indicated that greatest activity was directed against TV but Jasper and VR 299 were inactivated nearly as well by a typical serum preparation. Viral inhibition was slightly greater in the cyprinid (FHM) cell line than in the salmonid (CHSE-214) line. However, preparations of viruses that did not produce significant cytopathic effects (CPE) in FHM cells were comparatively insensitive to serum inhibition as determined in CHSE-214 cells. Buhl and Powder Mill were refractory to neutralization, as were VR 299 preparations without FHM-IPNV variants. Oyster virus (OV) was inhibited by normal trout serum, but to a considerably decreased extent compared to the Sp FHM-IPNV strain or to the serum-sensitive North American IPNV strains.
The multiplication and distribution of Pseudomonas anguilliseptica in artificially infected Japanese eels were studied by viable cell count. When the eels were injected intramuscularly with the lethal doses (107, 109/100 g fish weight) of a K antigen-possessing virulent strain at 12°C or 20°C, the viable cell numbers in blood and tissues decreased from 1 h to 12 h post-inoculation. This phase was followed by a static phase, in which the organism persisted at lower concentration or grew slowly, and the duration of this phase depended on the inoculated dosage and ambient water temperature. At the final phase, the organism appeared in rapidly increasing number and rose to high levels (108-10 viable cells/g or ml), which persisted until the death of fish. On the contrary, when inoculated with sublethal dose (105 CFU) of the virulent strain, with lethal dose of virulent strain at a higher water temperature(28°C), or with a dose of a K antigen-lacking avirulent strain, the organism disappeared completely from the tissues except the muscle of the inoculated site within 1-3 days. The final level of viable cell count in the moribund eels was higher than those of Aeromonas hydrophila or Vibrio anguillarum, and this difference was thought to reflect toxin production.
Four strains of Pseudomonas anguilliseptica isolated from pond-cultured Japanese eels, Anguilla japonica, in Taiwan were compared with Japanese and Scottish representative strains. From the results of agglutination and precipitation with rabbit antisera, and pathogenicity tests for Japanese eels, the three of Taiwanese isolates proved identical to Japanese K antigen-possessing type (K+ type) of P. anguilliseptica. The other Taiwanese strain seemed to be intermediate type between K+ and K- types.
The tissue location of Vibrio anguillarum bacterin in rainbow trout was studied using three delivery methods : intraperitoneal (IP) injection, immersion and per os. The fate of the bacterin was determined using histologically sectioned trout stained with anti-V. anguillarum (strain LS-174) fluorescein-labeled, purified, rabbit immunoglobulin G (IgG). The bacterin administered IP was accumulated by macrophages and observed in the kidney and spleen 28 d after injection. Bacterin delivered by immersion was observed on the surface of the gills, and in the gastrointestinal tract and kidney. Vaccine administered by the oral route was not detected systemically in test fish; the only accumulations found were in the lamina epithelialis of the lower intestine. Immunity developed with bacterins administered either by IP injection or immersion was considered to be systemic, because after vaccination, antigen was observed in a variety of internal fish tissues. Oral immunization apparently caused induction of a mucosal response because no antigen was observed in organs other than the intestine.
The direct fluorescent antibody technique (FAT) was successfully used to detect the causative agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, in Bouin's solution flexed and paraffinembedded egg and tissue sections. This method is superior to gram stain and may be particularly useful in detecting the BKD organism in fish with low-grade infection.