Investigations on the aerobic bacterial flora in the intestine of larval and juvenile stages in Japanese flounder (Paralichthys olivaceus) were carried out at two different fisheries stations. The bacterial flora of ambient water and feeds were also analyzed. The number of bacteria in the intestine of larvae measuring 10 mm in total length was 105 CFU/fish, remained nearly constant until 14 mm, and decreased to 104 CFU/fish in 15-19 mm. The decrease in bacterial numbers was hypothesized to be due to the change in feeds from live diets (rotifer and brine shrimp) to artificial diets. The bacterial numbers on ZoBell's 2216e agar were 104, 108, 105, and 106 CFU/ml or g in water, live feeds, artificial feed and minced fish, respectively. The composition of intestinal flora was characterized by two predominating groups of Vibrio and Pseudomonas followed by Moraxella, Cytophaga and Alcaligenes. Similar generic composition was recovered in water and live diets, while those of artificial diets comprised of Acinetobacter and Gram-positive bacteria in addition to Moraxella. Vibrio alginolyticus was frequently isolated from fish examined in one station where no apparent fish mortality occurred.
In this study the effect of immersion vaccination for Ayu vibriosis was investigated from the histopathological and immunohistochemical points of view, and the infection mechanism of vibriosis as well as the cause of death were proposed. Ayu, Plecoglossus altivelis, were vaccinated by the immersion in suspension of formalinkilled Vibrio anguillarum at concentration of 8.6 × 106 CFU/ml for 1 hour. The experimental fish were estimated 60 days after the vaccination. The water temperature was 20 to 22°C during the course of experiment. The cumulative mortality rate of the vaccinated group was lower by 40 % than the control group when being challenged with immersion for 10 minutes in suspension of V. anguillarum at concentration of 3.4 × 104CFU/ml, and by 20 % at concentration of 3.4 × 105 CFU/ml. In control group, lesions and bacterial proliferation were detected first in the skin, then in other organs through the whole body. Thus, the mechanism of infection was proposed as following. The bacterium first penetrated through the epidermis to the dermis where it proliferated and caused lesions. The bacterial invasion into, the blood stream through local capillary systems finally induced the systemic infection. The cause of death of the experimental fish was considered to be the heart and breath failure resulting from bacteremia. In the vaccinated group, when compared with the control, skin lesions were fewer in number and less severe in all individuals. When examined histologically, the skin lesions appeared later in the vaccinated group than the control, and a more extensive cellular infiltration with a reduced number of bacteria was observed in the former group. The results suggested that the vaccination strengthened the resistant properties of the epidermis and accelerated the cellular infiltration in the dermis of the host so that the bacterial penetration and proliferation were inhibited.
Six media commonly used for cultivation of Flexibacter columnaris were divided into two categories based on the presence or absence of certain salts. Growth was limited in Cytophaga, tryptone yeast extract or tryptone yeast infusion medium, none of which contain salts. Growth responses in Chase, Shieh and Liewes media containing salts provided improved cell yields and the Shieh and Liewes preparations were best. Comparisons of generation time were made between cells grown in Shieh and Liewes media. The generation time of F. columnaris, strain 238 grown in the Shieh broth was 147 min compared to 206 min in the Liewes broth. Glucose, pyruvate and citrate were not required for the cultivation of F. columnaris.
In order to further our understanding of the ecology and distribution of Vibrio anguillarum and its related organisms, we carried on a bacterial investigation in Otsuchi Bay from 1979 to 1981 where coho salmon (Oncorhynchus kisutch) were cultured. Fifty-eight strains out of 5337 isolates were identified as V. anguillarum. Thirty-six strains of the 58 strains were assumed to be originated from pen-cultured fish and were classified as serotype J-O-3. One strain, classified as J-O-1, was isolated from sea water far away from the rearing pen in Otsuchi Bay. The other twenty-one strains classified as J-O-8 were isolated from marine environments e.g. sea water, mud, plankton etc., all over the Otsuchi Bay.
In order to know the infection source of BMN in mass production of kuruma shrimp larvae, epizootiological investigations were undertaken on several incidents of the disease at hatcheries. Histological examination was made on the mid-gut glands of both wild mature female kuruma shrimp (18 cm in mean body length) used as spawners and cultured kuruma shrimp (5.6 cm in mean body length) which were the survivors of the larvae infected with the disease. The culturing water of the latter had been introduced into rearing tanks of larvae in which BMN epizootics occurred later. Epizootiological investigations suggested that latently infected spawners and cultured animals could become the vertical and the horizontal sources of infection, respectively. Histological examination demonstrated the nuclear hypertrophy (most characteristic cytopathological change of BMN) of the mid-gut gland epithelial cells in both latently infected females and cultured shrimp. Fluorescent antibody technique rapidly revealed the presence of BMN specific virus antigen in the hypertrophied nuclei of latently infected females.
A histopathological examination was made on red sea bream with a symptom of cloudiness on the body surface found in fish farms in Kyushyu in 1986. The examination revealed that the cloudiness was induced by the proliferation of prickle cells, the intra-and inter-cellular edema and over-secretion of mucus of the epidermis. The epidermis showed spongiosis caused by a severe inter-cellular edema. A highly inflammatory response was noted in the dermis. No virus-like particles were observed under an electron microscope either in the nucleus or in the cytoplasm of proliferating prickle cells. An enlargement of the respiratory epithelial cells of the gill as well as the fusion of gill lamellae and an increase of mucus cells were detected. From these histopathological findings on the skin and gills it was suspected that stimulative substances in the environmental water were part of the causes of the described disease.
Histopathological studies were conducted on diseased hirame (Paralichthys olivaceus) naturally infected with Rhabdovirus olivaceus (hirame rhabdovirus ; HRV) during 1984 and 1985 in Hyogo Prefecture and Hokkaido. Studies of hirame artificially infected with HRV (strain 8401-H) were also conducted. Signs of infection were congestion of the gonad, focal hemorrhage of skeletal muscle and fins, and accumulation of ascitic fluid. Histopathologically, the kidney indicated necrotic changes by nuclear degeneration of hematopoietic cells and hemorrage in the interstitial tissue. The spleen showed necrosis and hemorrhage in the pulp, and skeletal muscle revealed hyperemia and hemorrhage of capillary vessels. Hyperemia and hemorrhage were observed in the interstitial tissue of the seminiferous duct, ovarian lamella, and in the connective tissues around the seminal duct and oviduct of the testis and ovary. Mucosa of alimentary tract showed hyperemia and hemorrhage. No pathological changes were observed in the liver.
Hirame (Japanese flounder; Paralichtys olivaceus) wihch had been artificially infected(I. P. injection, 105.30TCID50/fish)withRhabdovirus olivaceus(hirame rhabdovirus; HRV)were reared at 5, 10, 15 and 20°C, and the cumulative mortality was observed. Moribund fish were employed for histophathological and hematological study. Cumulative mortality of test groups reared at 5, 10, 15 and 20°C were 40, 60, 20, and 0%, respectively. Moribund fish showed typical signs of HRV infection. The highest virus titer was obtained from the fish of the 5°C test group, followed by the 10°C test group. Histopathologically, the kidney of all fish reared at 5°C and moribund fish reared at 10°C indicated necrotic changes and hemorrage in the hematopoietic tissue. Necrosis was most severe in the 5°C test group. The spleen of the 5°C test group showed necrosis and hemorrhage in the pulp. Similar necrosis was also observed in the spleen of the 10°C test group, but was less severe. Infiltration of macrophages into the spleen of the 15°C test group, and hyperplasia of meranomacrophage in the tissue of the 15 and 20°C test group were noted. Hematologically, the antibody neutralizing activity against HRV of the sera was generally not very high, but the activity was comparatively higher in high water temperature test groups than that of low temperature groups. The increase of immature erythrocytes at 5°C, small lymphocytelike cells at 10 and 15°C, neutrophiles at 15°C and lymphocyte-like cells at 20°C were observed in the blood smeares.
The morphological characteristics of tumors which developed around the mouth of chum (Oncorhynchus keta) and masu salmon (O. masou) after surviving an experimental infection of Oncorhynchus mason virus (OMV) were studied histopathologically. The tumor tissues from the jaw of chum and masu salmon were composed of epithelial cells with the large light nuclei. The tumor was considered to be malignant epithelioma because of its active proliferation and intensive invasion of the connective tissues. Tumors which developed on the corneal epithelium and inneropercular of chum salmon showed similar characteristics to those around the mouth. Two types of tumor tissues also appeared in the kidney. One of them showed similar characteristics to those around the mouth and was presumed to be a transferable tumor. The others were characterized by their hyperplastic renal tuble cells (tuble epithelial cells) and their similarity in appearance to smooth muscle fibers.