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Enteric redmouth disease (ERM), which is caused by Yersinia ruckeri, has not been reported in Japan. In the present study, virulence of the bacterium were assessed by experimental chal­ lenges with the rainbow trout Oncorhynchus mykiss and 4 indigenous fish species in Japan: yamame (masu salmon parr) O. masou masou, amago O. masou macrostomus, whitespotted char Salvelinus leucomaenis and ayu Plecoglossus altivelis. When intraperitoneally (ip) injected with the bacterium at a dose of 7.1 x 10 2 CFU/g fish body weight (BW), rainbow trout, char, yamame, and amago showed the cumulative mortalities of 100%, 60%, 30% and 30%, respectively. The cumulative mortalities of ip­ injected ayu with doses of 1.5 x 10 6 , 1.5 X 10 5 , 1.5 X 10 4 and 1.5 x 10 3 CFU/g BW were 87%, 47%, 13% and 0%, respectively. In addition, the cumulative mortalities of ayu immersed in water containing 1.7 x 10 8 and 1.7 x 10 7 CFU/mL of the bacterium were 60% and 6.7%, respectively. Typical clinical signs of ERM, such as reddening of the wall of the oral cavity, tongue, upper jaw, lower jaw, operculum, bases of fins, or skin around the anus, were observed in all dead salmonids but not in ayu. Instead, exophthal­ mos, hemorrhage in the eyes, ascites or hypertrophy of the kidney were often observed in dead ayu. These results indicate that Yersinia ruckeri is a potentially dangerous bacterium to freshwater aquacul­ ture in Japan.

A recombinant fimbrial protein of Edwardsiella tarda was designed to detect the specific antibody in Japanese flounder Paralichthys olivaceus. The specific antibody was detected from the fish intraperitoneally injected with E. tarda expressing the fimbria on LB agar containing 3% NaCl, but not from those injected with E. tarda without the fimbria, Streptococcus iniae, S. parauberis or viral hemorrhagic septicemia virus. In a flounder farm suffered with edwardsiellosis, the antibody-positive fish increased in ration with the occurrence of mortality. These results suggested that E. tarda with the fimbria was important for epidemics of edwardsiellosis at flounder farms.

We evaluated the prevalence of the akoya oyster disease (AOD) in Japan and the pathogenicity of the unidentified causative agent. We obtained naive Japanese pearl oysters Pinctada fucata martensii from a site with no history of AOD. In separate experiments, the oysters were either challenged by intramuscular injection with the hemolymph of infected oysters or reared in several major pearl oyster farms throughout Japan. Both groups of oysters experienced high mortality and exhibited discoloration of the adductor muscle and histopathological changes characterizing AOD. Our data suggest that AOD is prevalent in pearl oyster culture fields throughout Japan and that the causative pathogen remains highly pathogenic to P. fucata martensii. When groups of oysters produced by repeated selective breeding against AOD were reared in different pearl oyster fields alongside uninfected naive oysters, the selectively bred oysters had significantly lower mortality than naive oysters. The recent decline in outbreaks of AOD can be attributed, in part, to the introduction of selectively bred oysters that are resistant to this disease.

The role of leukocytes from the kidney of Japanese flounder Paralichthys olivaceus in the encapsulation response was investigated using an in vitro model. The aggregation response of leukocytes to the target fish pathogen Ichthyophonus hoferi was observed over 12 h. The cellular aggregates mainly comprised granulocytes, which were characterized by intracellular peroxidase expression. Moreover, studies of isolated granulocytes demonstrated their adhesion to I. hoferi and formation of an initial layer in the encapsulation response. We observed that mRNA expression levels of three CC-chemokines (CCL3, CCL4 and CC-CLM) and one CXC-chemokine (IL-8) increased during the encapsulation process. Among these chemokines, CC-CLM and IL-8 were actively produced by granulocytes upon stimulation by I. hoferi. Flounder granulocytes therefore appeared to play an important role in the process by releasing specific chemokines upon pathogen recognition, thereby inducing subsequent cellular recruitment leading to encapsulation.

This study was made to clarify a possibility of vertical transmission of bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis. The presence of Flavobacterium psychrophilum on the surface and in the contents of unfertilized and fertilized eggs, which were obtained from ayu naturally carrying F. psychrophilum, was examined by a culture method. The bacterium was detected on the egg surface of 19 out of 65 unfertilized egg samples and of 5 out of 30 fertilized egg samples (300 eggs/sample). On the other hand, F. psychrophilum was not detected from the surface of eggs disinfected by povidone-iodine (5 ppm, 10 min) or hydrogen peroxide (150 ppm, 30 min). The bacterium was not detected in the contents of these surface-disinfected eggs. These results suggest that F. psychrophilum is not transmitted through intra-ovum infection in ayu eggs. Therefore, disinfection of eggs is useful to prevent the vertical transmission of BCWD in ayu hatcheries.

The effect of the addition of the aminoglycoside antibiotic tobramycin to the culture medium on selective isolation of Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, from ayu Plecoglossus altivelis was examined. The minimum inhibitory concentrations of tobramycin against all 37 strains ofF.psychrophilum tested were more than 6.3μg/mL. The addition of tobramycin to medium at a concentration of 5μg/mL was a better method for the isolation of F.psychrophilum, because the overgrowth of other bacteria was inhibited and the isolation rates of F.psychrophilum were improved. Especially, the use of tobramycin-supplemented medium could be effective in the examination of the external organs of carrier fish, in which viable bacterial counts of F.psychrophilum are lower than those of other bacteria.

Mass mortalities caused by akoya oyster disease have been occurring in cultured Japanese pearl oyster Pinctada fucata martensii in western Japan. Although the involvement of some causative agents was suggested, the cause of the disease is still unidentified. In this study, an experimental infection was performed by cohabitation of the diseased and healthy pearl oysters. A high mortality and characteristic disease conditions were observed in the test group. This result reconfirmed that this disease is caused by a certain infectious agent. To clarify the tissue distribution of the causative agent in diseased pearl oysters, two experimental infections were performed by transplantation of pieces of various tissues or injection of homogenates of various tissues from diseased pearl oysters into healthy pearl oysters. The hemolymph, mantle and adductor muscle showed high infectious titers compared with those of the heart, digestive gland and hemocytes. The mantle had the highest infectious titer. Moreover, injection of the supernatant of a mantle homogenate also transmitted the disease to test pearl oysters. These results suggest that the causative agent is concentrated in the mantle.

Development and growth of the monogenean Neoheterobothrium hirame on Japanese flounder Paralichthys olivaceus were investigated at 15°C, 20°C and 25°C, and production of antibodies against N. hirame was monitored in relation to the development of the parasite. After settlement on the gill filaments, hematin cells in the gut wall of N. hirame were first observed on Day 17, Day 10 and Day 7 post-exposure (PE) of oncomiracidia at 15°C, 20°C and 25°C, respectively. The parasite started to move to the buccal cavity wall from Day 38, Day 24 and Day 24 PE and eggs were observed in the uterus from Day 59, Day 38 and Day 31 PE at each temperature, respectively. Intensity of infection decreased subsequently and infection persisted until Day 122, Day 66 and Day 52 PE, respectively. Antibody titers gradually increased from Day 52 PE at 15°C, while at 20°C and at 25°C, they rapidly increased after a decrease in the intensity and prevalence of the infections was observed. This suggests that the death of the parasite induces a host reaction, resulting in the production of antibodies against N. hirame.

To clarify the causative agent of the anemia that has been recently prevailing in wild and cultured Japanese flounder Paralichthys olivaceus, anemic flounder were generated by repeated bleedings and examined hematologically. When flounder were bled once a week at 0.5 or 1.0% (volume/body weight) for 12 weeks without feeding, severe anemia developed in the fish.The anemia showed significant reductions in the hemoglobin concentration, mean corpuscular cell volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration, and structural anomalies of erythrocytes occurred such as low hemoglobin accumulation in the cytoplasm and a deformed outline. The hematological characteristics of the anemia caused by the repeated bleedings was almost identical to the anemia observed in wild Japanese flounder, supporting the previous conclusion that the blood-feeding activity of the monogenean Neoheterobothrium hirame is the cause of the anemia. Even after the last bleeding, it required 8 weeks for the anemic fish to recover in a feeding condition. The long period needed for the recovery also supports the previous conclusion that the anemic wild fish without N. hirame can be recognized as fish in the recovering phase from the anemia.

Edwardsiella tarda causes high mortality infections in fish. To determine the efficacy of a vaccine against E. tarda, a formalin-killed preparation of atypical E. tarda FPC503 was intraperitoneally injected into red sea bream Pagrus major with or without the oil adjuvant, Montanide ISA 763 A VG. Both vaccine regimens showed protective efficacy against experimental infection with E. tarda FPC503 at 28 and 56 days post-vaccination. An enzyme-linked immunosorbent assay (ELISA) revealed the specific antibody production in vaccinated fish but the relationship between protective efficacy and antibody titer was not clear. The present results indicate that the formalin-killed vaccine is useful for the prevention of atypical E. tarda infection in red sea bream.

近年ヒラメに頻発している原因不明の貧血症について, 血液学的, 寄生虫学的検査と造血器官の病理組織学的観察を行った。血液学的には, 幼若赤血球の増加と赤血球細胞質の空胞変性および染色性の低下が認められた。病魚の造血器官にヘモジデリンの沈着や組織壊死像はほとんど見られず, 貧血魚にはN.hirameが高率に寄生していた。このことから, 本貧血症は寄生虫による失血性貧血であることが強く示唆された。

The characterization of rainbow trout peripheral blood leucocytes (PBL) was carried out by flow cytometry (FCM) analysis and by transmission electron microscopy. Two major cell populations were identified in FCM analysis. One population showed small values in both forward scatter (FS) and side scatter (SS), and the other population showed large values in both parameters. These major populations were divided into two sub-regions each (A/B and C/D). There was no significant difference in cell populations between sub-regions A and B. However, a difference was observed in the small and large lymphocyte percentages between sub-regions C and D. The majority of regions (A and B) were constituted by neutrophils (56% and 66%, respectively), and monocytes accounted for 9% and 7% of the cells, respectively. Large and small lymphocytes were also present in these regions, accounting for 26% and 6% of the cells in sub-region A and 14% and 7% in sub-region B, respectively. In sub-regions C and D, cells were mainly constituted by large and small lymphocytes (33% and 49% in C, and 7% and 70% in D, respectively) and thrombocytes (14% and 23%).

Hematological characteristics of the anemia prevailing in wild Japanese flounder Paralichthys olivaceus were examined in detail by cell counting and microscopy. The erythrocyte counts, MCH, and MCHC decreased correlatively with the decrease in the hemoglobin concentration. Moreover, the reduction of erythrocyte volume was also observed in many anemic fish. Light microscopy showed erythrocytes with structural abnormalities, such as vacuolated, weakly stained cytoplasm and deformed outlines. In the TEM observations of erythrocytes, an insufficient accumulation of hemoglobin was observed as well. These results indicate that the anemia can be identified as a hypochromic microcytic anemia accompanied with hypoglobulia and a structural abnormality of the erythrocytes.

プラスチックシャーレの底に Edwardsiella tarda を付着させて作った細菌薄層にコイおよびニジマスの血液をのせ, 薄層上に形成された食菌プラークを画像処理し, プラークの数および面積を測定した。10, 20, 30℃の反応温度における貧食能を比較した結果, コイでは30℃で最大の貧食能を示したが, ニジマスではプラーク数および全プラークの面積は20℃で, 1個当りのプラーク面積は30℃で最大であった。