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Two-year-old tiger puffer Takifugu rubripes, persistently infected with the monogenean Heterobothrium okamotoi for longer than one year, were cohabitated in an aquarium with one-year-old tigerpuffer with no previous record of infection (naive fish) for 70 days. The infection level in the naive fish rose sharply, and the intensity of infection reached its highest on day 30 (estimated 16, 700 parasites on the gills per fish). In the persistently infected fish, on the other hand, no change was recorded in the infection level, which was much lower than that of the naive fish. Based on the compositions of parasite developmental stages in the two fish groups, we hypothesize that the protection mechanism found among the persistently infected fish functioned in at least three occasions : firstly when the oncomiracidium settled on the gills, secondly when the parasite developed from a worm without clamps to one with a pair of clamps, and thirdly when it migrated and grew on the branchial cavity wall. The persistently infected fish produced anti-Heterobothrium antibodies, while no antibody was detected in the naive fish. These results suggest that a persistent infection induces protection against H. okamotoi, but the factors responsible for effective prevention against reinfection remain to be clarified.

The dynamics of development and production of Thelohanellus hovorkai (Myxozoa) were examined to investigate factors inducing haemorrhagic thelohanellosis in carp, Cyprinus carpio L. Fresh actinospores of T. hovorkai were harvested from the oligochaete alternate host, Branchiura sowerbyi, and used for infection experiments with myxosporean-free carp. Visualization of actinospores by fluorescent labelling revealed that sporoplasms penetrated the gill filaments of carp immersed in an actinospore suspension as early as 30 min post-exposure (PE). Plasmodia of T. hovorkai developed in the connective tissues of various organs and matured 3-5 weeks PE; dispersion of myxospores from degenerate plasmodia occurred 5-7 weeks PE. Challenges with a high dose of actinospores (4.5 x 10(6) spores per fish) resulted in the onset of disease, which was more easily achieved by the oral intubation of actinospores than by immersion in an actinospore suspension. Actinosporean-free B. sowerbyi were exposed to different densities of myxospores (10(4)-10(6) spores per oligochaete) and subsequently reared at different temperatures (15, 20, 25 degrees C). At 20 and 25 degrees C, actinospore releases were first detected 40-43 days PE, with multiple peaks of release (max. 7 x 10(5) actinospores day(-1)) during the next 60 days. We concluded that the developmental cycle of T. hovorkai was completed within 3-5 months at 20-25 degrees C, and that the ingestion of large numbers of actinospores orally, possibly by feeding on infected oligochaetes, resulted in a disease condition in carp.

Attachment-inducing capacities of skin epithelial extracts of yellowtail, Japanese flounder and red sea bream on oncomiracidia of the monogenean Benedenia seriolae were examined. Clear differences were not detected in the capacity among the fish species, although B. seriolae infects only yellowtail and its congeners in Seriola. This suggests that either the capacity is not host specific or host-specific attachment-inducing capacity cannot be detected by the assay method. Further, the attachment-inducing capacities were suppressed by wheat-germ lectin and concanavalin A in skin epithelial extracts of Japanese flounder and yellowtail, respectively. This suggests that some sugar-related chemical substances existing in fish epithelia induce the attachment of B. seriolae oncomiracidia.

Pathological changes in cultured tiger puffer, Takifugu rubripes, with emaciation disease in Kyushu, Japan were studied histologically. In most cases, diseased fish were heavily infected with at least one of three myxosporeans (Myxidium fugu, Myxidium sp. and Leptotheca fugu) and two unidentified hyperparasitic microsporeans, attached to, or in, the intestinal epithelium. Myxidium fugu attached to the surface of the epithelium, caused no noticeable effects on the host tissue, irrespective of its infection with the hyperparasite. Myxidium sp., which proliferated in the epithelium, induced severe pathological changes including accumulation of cell debris between the epithelium and lamina propria and resultant detachment of the epithelium. Leptotheca fugu, another histozoic myxosporean, induced degeneration of the epithelium, associated with massive infiltration of macrophages into the epithelium to encapsulate parasites. When L. fugu was infected with its hyperparasitic microsporean, shortened villi were also observed. This is probably because passage of macrophage-parasite aggregates through the basement membrane of the epithelium severely damaged the epithelial structure. It is evident histologically that, unlike epicellular M. fugu, histozoic Myxidium sp. and L. fugu with or without hyperparasitic microsporeans, were highly pathogenic to host fish. This strongly suggests that they are causative agents of the emaciation disease.

魚類の感染症(伝染病)は,養殖生産や種苗生産の発展と表裏して増加を続けている。実態把握の難しい天然水域における魚類感染症についても,環境汚染や種苗放流など人間生活に伴う悪影響が懸念されている。予防接種(ワクチネーション)は魚類にも有効であり,数種の魚類感染症に対するワクチンが実用に共されている。また,最近,「水産資源保護法」の改正と「持続的養殖生産確保法」の制定によって,日本では初めて魚類防疫制度が発足した。しかし,予防接種にも防疫制度にも多くの課題が残されており,魚類感染症の防除対策に関する幅広い研究と技術開発が期待される。

The intracellular ovarian parasite causing unaesthetic appearance in the ovary of Pacific oysters Crassostrea gigas in Japan has remained unidentified since the first report of its occurrence in the 1930s.The parasite was observed through transmission electron microscopy, and identified as the paramyxean Marteilioides chungmuensis, which was first reported from Pacific oysters in Korea.In order to obtain information on the development of the parasite, the diameters of both the parasite and the infected ova were measured.The growth of the parasite was highly correlated with the growth and maturation of host gonad cells.Histological observations suggest that M. chungmuensis invades immature ova, which move to the center of the follicle along with its development, and infected ova are finally released outside through the genital canal.

We report green fluorescent protein (GFP) as a useful reporter molecule to track the occurrence of Pseudomonas plecoglossicida in ayu Plecoglossue altivelis. The gfp gene was placed in a shuttle vector pME4510 and then three kinds of GFP vectors, pSKL01, pSKT03 and pSKN04 were constructed, in which lac, tac, and neophosphotransferase II (npt2) promoters drove the expression of GFP, respectively. A promoterless GFP vector was designated as pSKP02. P. plecoglossicida carrying pSKT03 gave the highest expression of GFP. In addition, pSKT03 was relatively stable under non-selective pressure and there was no significant changes in the in vitro growth and pathogenicity of the cells carrying this plasmid. The cells of fluorescent P. plecoglossicida attaching to the body surface of ayu were easily detected under a fluorescence microscope. It was revealed that they adhered predominantly to the microscopic injuries in the skin and fins.

Seasonal occurrences of the abnormally enlarged ovary of Pacific oystersCrassostrea gigascaused by an unidentified intracellular protozoan parasite was investigated in Gokasho Bay, Mie Prefecture, Japan. Cultured and wild oysters were collected bimonthly for one year from July 1996, and seasonal fluctuations in the prevalence of infection, parasite development and histological changes of the infected tissue were examined. Parasites, very similar toMarteilioides chungmuensisinfecting Pacific oysters in Korea, had 8 forms, 4 of which were frequently observed, while the other 4 forms, which were not described inM. chungmuensis, were rare. The total prevalence of infection (percentage of infected oysters positive for at least one of the two examinations : gross and smear inspections of the ovary) was usually higher than the gross prevalence of infection, indicating that infected oysters included ones without visible legions. In cultured oysters, the prevalence of infection increased in summer, suggesting that active multiplication of the parasite takes place in warm water months. From autumn through spring, no substantial change in the total prevalence of infection was observed in cultured oysters (18-20%), while it continued to decline in wild oysters during that period. These differences between cultured and wild oysters could be attributed to the oyster size and environmental factors such as salinity. Histologically, the number of infected oocytes increased in summer, but decreased in winter. From the variations in the parasite morphology and maturation of oocytes, the life cycle of the parasite and the mechanism of the legion formation are postulated.

Early stages of Glugea plecoglossi (Microspora) are difficult to detect by normal light microscopy. In situ hybridization (ISH) protocol with DIG-labeled oligonucleotide probes designed from SSUrRNA of G. plecoglossi was established with the aim of detecting all stages. All developmental stages of G. plecoglossi in xenomas could be detected by ISH. First, we aimed to evaluate the suitability of rainbow trout as an experimental animal in place of ayu for G. plecoglossi. Immersion challenge tests showed that the susceptibility and location of G. plecoglossi did not differ between ayu and rainbow trout. Cyst forming sites were different among challenged trout by the three methods (intubation, immersion and intraperitoneal injection). In the intestinal epithelium of rainbow trout (Oncorhynchus mykiss) at 5 min post-intubation of G. plecoglossi spores, we found possible initial stages, which were positive in ISH and negative in Uvitex 2B staining. This finding suggests that G. plecoglossi spores discharging the polar tube pierced the gut epithelial cells, and injected the infective sporoplasm. The present study showed that ISH is a promising tool for studying transmission modes of G. plecoglossi in fish and that rainbow trout is a useful replacement host to identify the pathobiological characteristics of G. plecoglossi.

Fertilized coho salmonOncorhynchus kisutcheggs were immersed in diluted broth culture of Flavobacterium psychrophilum(1.0×108, 1.0t106, 1.0×104CFU/mL) for 30 min before water-hardening, after water-hardening and at eyed-egg stages. Just after exposure to the bacteria, the eggs were disinfected with povidone-iodine, and then discretely incubated in flowing water. After 7-28 days of incubation, F. psychrophilumwas isolated only from the egg group which had been exposed to 1.0 t 108CFU/mL before water-hardening. No bacteria were detected from the other egg groups exposed to 1.0 t 106or 1.0 t 104CFU/mL as well as the groups exposed to 1.0 t 108 CFU/mL after water-hardening or the group at the eyed-egg stage. The viable counts ofF. psychrophilumin the contents of the infected eggs ranged from 103to 107 CFU/g. Observations on the frozen sections stained by indirect immunofluorescent antibody technique revealed that manyF. psychrophilumcells were located within the infected eggs at the eyed stage. It was concluded that F.psychrophilumentered the eggs during the water-hardening stage.

Polymerase chain reaction (PCR) and indirect fluorescent antibody technique (IFAT) were developed for the detection of Kudoa amamiensis (Multivalvulida : Myxozoa), which produces pseudocysts in the skeletal musculature of yellowtail Seriola quinqueradiata. A nested PCR derived from the 18SrDNA sequences of K. amamiensis proved to be highly specific and sensitive, detecting a single spore in a 20μL reaction mixture. In a periodic monitoring of yellowtail, the single-round and nested PCRs were conducted as well as conventional diagnostic techniques (visual inspection, wet-mount observation and histological examination). After one month culture of yellowtail in an endemic area, neither pseudocysts nor spores could be detected visually, whereas 53% of the examined fish were positive for infection by the nested PCR. After 3 and 5 months, macroscopic pseudocysts were detected in 27% and 60% of the fish, respectively, while the nested PCR was more sensitive (93% and 70% were positive, respectively). Histologically, intracellular organisms with several internal cells were detected only in the muscle fibers of the samples which were positive by PCR tests. By the IFAT using a rabbit antiserum to sonicated K.amamiensis spores, both pre-spore and spore stages reacted, and intracellular plasmodia were positively identified as presporogonic stages of K. amamiensis. In conclusion, we developed the IFAT and the nested PCR for the early diagnosis of K. amamiensis, and described for the first time its intracellular presporogonic stage in the muscle fiber of yellowtail.

Three myxosporeans and 2 hyperparasitic microsporeans were found in the intestine of emaciated cultured tiger puffer Takifugu rubripes in Kyushu district of Japan. Morphology of the 3 myxosporeans were examined and described as Myxidium fugu n. sp., Leptotheca fugu n. sp. and unidentified Myxidium sp. Spores of M. fugu n. sp. having 2 club-shaped polar capsules were bean-shaped, 14.0 (13.5-15.5) μm in length and 9.0 (8.0-10.0) μm in width. Spores of L. fugu n.sp. having 2 spherical polar capsules were trapezium-shaped, 9.0 (8.3-9.5) μm in length and 14.0 (13.0-15.0) μm in thickness. Their piscine intestinal developmental stages were illustrated and arranged in a hypothetical developmental sequence. Plasmodia of M.fugu n. sp. were located on the epithelium of intestine, whereas those of L. fugu n. sp. and Myxidium sp. were found intercellularly in the intestinal epithelium. Only a few mature spores could be observed in these myxosporeans, but the differences in stainability by Diff-Quik and Uvitex 2B and in location of development made it possible to differentiate the species. Two hyperparasitic microsporeans were often found in the plasmodia of M. fugu n. sp. and L. fugu n. sp, but their pathogenic effects to host myxosporeans were unclarified.

To elucidate the process of the uptake of suspended particles into the skin, fins and gills of rainbow troutOncorhynchus mykiss, 100 fingerlings were immersed in a suspension of 1μm diameter fluorescent microspheres and rinsed with water. The fate of microspheres adhering to body surface was traced for elapsed times ranging from 1 min to 24 h. Although most microspheres were removed in a 2 h-rinse with water, a few were observed by light microscopy to be located in both epidermal and dermal tissues which had microscopic injuries. As observed by electron microscopy, microspheres were taken up by migrating epithelial cells, or embedded in the dermis covered with these cells. Some of those microspheres embedded in dermis were endocytosed by macrophages. Such features were confirmed in artificial wound experiments. We conclude that microscopic injuries to trout could be an important route for the uptake of antigen administered by immersion, 2 to 24 h after fish suffer from injuries.

Based on the nucleotide sequences of 16S rDNA, Japanese isolates of atypicalAeromonas salmonicidafrom different fish species can be divided into four groups; isolates from each species of fish (including fish reared in similar culture conditions) always belonged to the same group. Isolates from goldfishCarassius auratushad sequences identical to those from goldfish in the USA and formed the first group. Isolates from Japanese eelAnguilla japonica and marine fishes (greenlingHexagrammos otakii, Japanese flounderParalichthys olivaceus, shotted halibutEopsetta grigorjewi and Schlegel's black rockfish Sebastes schlegeli) formed the second group, and had identical sequences with those ofA. salmonicidasubsp.achromogenes JCM7875T, A. salmonicida subsp. masoucida JCM7873TandA. salmonicida subsp. smithia ATCC49393T. The third and fourth groups composed of isolates from coloured carpCyprinus carpioand common carpC. carpio, respectively. This indicates that the expansion of ulcerative disease in recent years is caused by strains which have phyletic lines different from goldfish isolates.

1999年5月から12月に新潟県海川で天然遡上アユ, 標識放流したアユ種苗, 流下アユ仔魚および他の生息魚を採集して F.psychrophilum の検出を試みた。 IFAT または nested PCR により検査した結果, 放流前のアユ種苗の一部, 採集された全てのグループのアユと他の4魚種（ヤマメ, ウグイ, ウキゴリ, ヨシノボリ）および成熟アユの卵から本菌が検出され, また, 河床の微細藻類や河川水の一部が PCR 陽性を示した。 これらの結果から親魚から仔魚への冷水病菌の伝播, 放流魚や他魚種等から天然遡上アユへの伝播の可能性が示された。

Genetic variability among strains of Flavobacterium columnare (formerly Flexibacter columnaris) was characterized using restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, 16S rDNA sequencing, and DNA-DNA hybridization. A total of 23 Fl. columnare strains isolated from diseased fishes in various areas were divided into three distinct genomic groups. The main group, genomovar 1 consisted of 20 strains, including the type strain. Two strains were assigned to genomovar 2, and one to genomovar 3. However, as we found their phenotypic characteristics to be identical, there is no evidence to classify strains of these genomovars as belonging to new species or sub-species.

1996年ごろからニシキゴイおよびマゴイ養殖場に皮膚の潰瘍病変を特徴とする疾病が流行し, 病魚から A.salmonicida と思われる菌株が分離された。ニシキゴイ由来菌株はカタラーゼ陰性, マゴイ由来菌株はオキシダーゼ陰性という特異的な性状を示したが, その他の性状および 16S rRNA 遺伝子の解析により, いずれの菌株も非定型 A.salmonicida に分類された。マゴイに対する皮下注射では, 両者とも強い病原性を示し, また自然発病魚に似た穴あき症状を呈した魚も認められた。

To determine whether suspended particles adhere to fish, fluorescent latex microspheres, 1μm in diameter, were experimentally administered to rainbow trout fingerlings by bath immersion for 5 minutes. After vital staining in a 0.05% trypan blue solution for 10 minutes, microspheres in the skin and fins were observed in the stained area in a patchy distribution.Histologically, these microspheres were located on the surface of microscopic injuries. It was confirmed by artificial wound experiments that microspheres adhered to the surface of such wounds. By electron microscopy, microspheres were found to adhere to swollen superficial epithelial cells or collagenous matrixes which lack cuticular secretion. In the gills, however, no area stained with trypan blue, but a cluster of microspheres associated with mucus and debris was lodged between the lamellae. We consider that cuticular secretion could prevent particles from adhering to the epidermis of fish.

Eggs of coho salmon (Oncorhynchus kisutch), rainbow trout (O. mykiss), and masu salmon (O. masou) were artificially inseminated and subsequently immersed in a modified Ordal and Anacker's broth culture of Cytophaga psychrophila (107-108CFU/ml) for 30min. The bacterium was recovered from the eggs at the eyed stage with viable cell counts ranging from 105 to 107CFU/g. The fertilized eggs were disinfected with 50ppm povidoneiodine for 15min immediately after experimental infection. However, C. psychrophila was detected by a broth culture method from 60-80 percent of the surface-disinfected eggs. At the eyed stage, the eggs were disinfected again with povidone-iodine at concentrations of 50-1, 000ppm for 15-120min. C. psychrophila was isolated from all of them at densities of 104-107CFU/g. The uninfected control eggs were all negative for the bacterium. It is concluded that the povidone-iodine treatment is ineffective to eliminate C. psychrophila from salmonid eggs.

A new epizootic causing mass mortalities among cultured Japanese abalone, Sulculus diversicolor supratexta, occurred in Kanagawa Prefecture, Japan, during the period from June to October in 1997. Diseased abalone lost the ability to adhere and eventually died on the bottom of aquaria. White spots on the foot were a characteristic sign of the disease. In sections of tissues with lesions, there was necrotic degeneration in muscle fibers, and homogeneous bacterial cells were isolated from the infected tissue. Isolates of this bacterium were Gramnegative, facultatively anaerobic, rod shaped organisms that were motile by a single polar flagellum. They were identified as Vibrio carchariae on the basis of phenotypic characteristics, 16S rRNA gene sequences and DNA-DNA hybridization, although some phenotypic properties of the isolates differed from those of references. Infection experiments confirmed the pathogenicity of the bacterium to Japanese abalone. This is the first report of the isolation of V. carchariae from a diseased shellfish.

Hemorrhagic thelohanellosis in two varieties of carp (Cyprinus carpio) caused byThelohanellus hovorkai was investigated by experimental infection. One-year old coloured carp (koi-carp) and common carp were kept for five weeks in tanks with oligochaetes (Branchiura sowerbyi) collected from a pond where hemorrhagic thelohanellosis was enzootic. Another group of coloured carp was exposed only to the effluent from a tank containingB.sowerbyi. Four weeks post-exposure, both coloured carp and common carp maintained withB. sowerbyishowed clinical signs of infection and chronic mortalities. On the other hand, neither mortalities nor clinical signs were observed in the effluent group, though light infections withT. hovorkaiwere detected. Control groups of coloured carp and common carp maintained in clean tanks showed no signs of infection at all. Through a standardized trypsin digestion method, the intensity of infection withT. hovorkaiin carp maintained withB. sowerbyiwas determined to be more than 50, 000 spores/g-tissue. Histological observations revealed thatT. hovorkaiplasmodia were most frequently detected in connective tissues of the intestine and the ventral skin in the cohabitation group, whereas they were found most frequently in the gills of the fish of the effluent group. In conclusion, hemorrhagic thelohanellosis was experimentally induced in carp by maintaining hosts withB. sowerbyiregardless of host's varietal difference, and the ingestion ofB. sowerbyiinfected with the actinosporean may be an important factor inducing the disease conditions.

Enzyme electrophoresis for superoxide dismutase (SOD) and catalase divided 144 Edwardsiella tarda strains into two groups; group 1 contained all the strains isolated from diseased fish in Japan and group 2 included 25 strains from non-disease sources and reference strain ATCC15947T. These groups could be similarly distinguished by a simple catalase test; group 1 showed an instantaneous bubbling, whereas in group 2 bubbling was delayed or weak. PCR-RFLP analysis usingHhaI revealed that the two groups differed in their 16S rDNA genotypes. Furthermore, this analysis subdivided group 1 into two genotypes.

A vital staining technique with fluorescein diacetate (FDA) and propidium iodide (PI) was used for determination of viability of myxosporean stage spores of Myxobolus artus and actinosporean stage spores of M. cultus. Viable spores stained green with FDA and non-viable spores stained red with PI were clearly distinguishable in M. artus myxosporean spores released from live infected fish, while the spores collected from pseudocysts were not well stained with FDA. Immediately after release from the oligochaete, Branchiura sowerbyi, actinosporean spores of M. cultus, stained bright green in the sporoplasms and red in the spore processes. In vitro survivability tests revealed that the longevity of M. artus spores was 5 months at 25°C and 15 months at 18°C. Drying and ultraviolet irradiation (36 000 mW s cm–2 for M. artus myxosporean spores, 600 mW s cm–2 for M. cultus actinosporean spores) were most effective as sporicidal treatments.

The nested PCR was used to detect Cytophaga psychrophila carried by juvenile ayu (Plecoglossus altivelis) and eyed eggs of coho salmon (Oncorhynchus kistch). Ayu juveniles were caught in Lake Biwa and frozen shortly after landed. The coho salmon eggs from the USA and Japan were collected on a lot-by-lot basis at Narita airport or hatcheries when the containers arrived. Chelex 100 was used as a medium for extraction of DNA from these samples. Utilizing the PCR, a specific primer pair of PSY1 and PSY2 for C. psychrophila was used to amplify its 16S rDNA segments. An amplification product of the expected size was obtained from 5 of 24 DNA extractions from the kidney of ayu samples in April 1995. These fish had been previously diagnosed as negative for the presence of C. psychrophila by cultivation on TYE agar and IFAT. Five of 11 lots of coho salmon eggs were found to be positive for C. psychrophila by PCR. Four of these five lots had also been diagnosed as positive by cultivation and IFAT. In addition, the sensitivity of the PCR amplification for detecting C. psychrophila in kidney samples of ayu was calculated to be 1.5 CFU per PCR reaction tube.

The possibility of antibody-mediated effect of elevated water temperature treatment of Glugea-infected ayu was investigated by challenge test and ELISA. The treatment effectively reduced the prevalence and intensity of infection. In naturally infected fish, the prevalence of infection was significantly decreased by elevated water temperature treatment. The treatment was more effective in naturally infected fish treated thrice than those treated twice, as demonstrated by the smaller number of cysts in the former group of fish than in the latter. The antibody levels were also significantly increased by the treatment in naturally infected fish. However, in the experiment using unifected fish, no significant increase in the antibody levels by the treatment was seen, though the prevalence of infection was significantly decreased by the treatment. Through the challenge test, it was clear that the effect was temporary, and protective immunity was not confirmed by the treatment. These results suggest that the effect may not be antibody-mediated. Thus it is recommended that the elevated water temperature treatment be repeated and applied with continuous monitoring of reinfection.

An invesigation on optimal temperature for the growth and protease production of a mesophilic bacterium, Aeromonas hydrophila was carried out. The studied 12 strains of fish origin were collected and stored at -80deg;C. Optimal growth temperature was determined by a temperature gradient incubator using TYE medium, a suitable medium for protease production. The protease production was estimated by the Bio-Rad DC protein assay with broth culture supernatant at around stationary phase. Casein (Hammarsten quality) was used as a protein substrate. There was no considerable amount of protease within log phase culture. Although the optimum temperatures for growth and protease production were variable with the strain, optimal temperatures for protease production were always lower than those for the growth. Peak growth was favoured at 34.5±1.0deg;C, while the protease production at 27.6±4.9deg;C. The differences among the peaks of protease production and growth were 1 to 15deg;C.

Seasonal occurrence of Myxobolus artus infection in common carp Cyprinus carpio was monitored in a fry pond (Tokyo Metropolis) for one and a half year. Formation of pseudocysts in the skeletal muscle was detected from August, and the prevalence of infection reached a maximum of 10% in September. After maturation of pseudocysts, diseased fish released spores into the water at a maximum level of about 3t106 spores/ day/fish in October. Spore release decreased in late autumn and winter, increased in spring and exhausted in the following summer in 1-year old recovered fish. Mortality of diseased fish was negligible in September. However, chronic mortality with a typical sign of anemic gills was found during the spore releasing period. During a high level period of spore discharge, hematological indices (Ht, Hb, and RBC) in diseased fish significantly decreased, and the percentage of immature erythrocytes increased. Histopathological examination showed that numerous phagocytosed spores caused local occlusion and destruction of gill capillaries and exfoliation of gill epithelium, suggesting blood loss from the damaged gills. It was suggested that chronic hemorrhagic anemia was associated with spore discharge from carp heavily infected with M. artus.

The humoral immune response of ayu (Plecoglossus altivelis) to the microsporidian parasite Glugea plecoglossi was investigated. The antibody levels of sera from ayu, to which G. plecoglossi spores were administered orally, were measured by indirct ELISA. While the cysts appeared from 27 D.P.I. (days post-infection), the antibodies against the intact spores were first detected at 10 D.P.I. and no more significant increases in the antibody levels were observed. The uptake of spores through the gut epithelium was histologically observed. Additionally, sera from ayu in a cultured stock which showed 50% prevalence of infection were obtained and the antibody levels were measured by the ELISA. The antibody level was variable but no important difference was observed between fish with or without cysts. The intensity of infection (number of cysts) was not related to the antibody level both in the artificially and naturally-infected fish. These results suggest that the antibody production against the intact spores of G. plecoglossi plays no protective role against G. plecoglossi infection.

On the basis of the 16S rRNA sequence data analysis among the closely related species, the specific primers for Flexibacter maritimus, Flavobacterium branchiophilum, and Cytophaga columnaris were constructed. The specificity in amplifying the 16S rDNA of each species was confirmed by using selected strains of related bacteria and principal fish pathogenic bacteria. A primer pair of MAR 1 and MAR2 could differentiate F. maritimus from other species. The PCR performed with a pair of primers specific to F. branchiophilum failed to amplify the 16S rDNA. However, the PCR with a pair of a specific primer BRA1 and a universal primer 1500R succeeded in specifically amplifying 16S rDNA from F. branchiophilum. The Nucleotide Sequence Database (GenBank) contains two different 16S rRNA sequence data for C. columnaris. On the basis of these data, we produced two primer pairs. A pair of COL1 and 1500R amplified the 16S rDNA from 5 of 7 strains of C. columnaris, and a pair of COLa and COLb worked for the rest of the strains. In addition, these two groups within C. columnaris were supported by PCR-RFLP.

The monogenean Benedenia epinepheli (Yamaguti, 1937) Meserve, 1938 is redescribed and reported for the first time from the fins, eyes, and body surface of cultured marine fishes of Japan. Because of close similarity in the general morphology of the reproductive system and median sclerites, comparable morphometrics, and the presence of a flap slightly left of the common genital aperture, Benedeniella congeri Yamaguti, 1958 (= Neobenedeniella congeri (Yamaguti, 1958); Benedenia congeri (Yamaguti, 1958)) and Benedenia sp. of Tsutsumi and Ito, 1965 are synonimized with this species. Twelve new hosts are presented, bringing the total number of known hosts to 25 (11 Tetraodontiformes, 9 Perciformes, 3 Scorpaeniformes, 1 Pleuronectiformes, and 1 Anguilliformes). The ability of B. epinepheli to cause death of cultured fishes during heavy infection, and its low host specificity, ubiquitous distribution in western Japan, and the high susceptibility of marine aquarium fishes are indicative of the parasite's great potential to become a disease problem.

Under laboratory conditions at 25°C, the monogenean Neobenedenia girellae (Hargis, 1955) rapidly developed on Japanese flounder (Paralichthys olivaceus), and it took 15-17 days from egg to maturation.The whole process of egg formation from the release of oocytes from the germiduct to laying of egg capsule required a mean of 73sec. At room temperatures between 27-30°C, large adults laid a mean of 35.4eggs/h, while smaller ones a mean of 12.2eggs/h. Eggs began to hatch after incubation for 5-6 days at 25°C with natural cycle of illumination, but no hatching occurred at 15°C. It took 10-11 days to reach sexual maturity where the minimum body size for an egg-laying adult was 2.1mm. When compared with other benedeniine species, N.girellae required a shorter time and a smaller body size to attain maturity and showed a high level of egg output. One-day old larvae were found only on the fins of Japanese flounder.As the parasites grew, they were found not only on the fins but most commonly on the dorsal and ventral body surface of fish, suggesting migration of the parasite from the fins.

Japanese eel (Anguilla japonica) neutrophils underwent a striking increase in oxygen consumption by stimulation, which is usually called respiratory burst. This increased uptake of oxygen was insensitive to mitochondrial respiration inhibitor such as potassium cyanide. Superoxide was detected during the respiratory burst by measuring chemiluminescence (CL) enhanced by 2-methyl-6-phenyl-3, 7-dihydroimidazo [1, 2-a] pyrazine-3-one (CLA) which is highly specific and sensitive to superoxide. EDTA blocked while EGTA stilumated CLA-enhanced CL. These characters of the respiratory burst suggest that Japanese eel neutrophils have NADPH oxidase-like activity similar to the mammalian NADPH oxidase. Sodium azide, an inhibitor of myeloperoxidase (MPO) activity, did not affect luminol-enhanced CL, which is thought to be dependent on hypochlorous acid (HOCl) produced by catalysis of MPO. It is suggested that Japanese eel neutrophils do not generate HOCl during the respiratory burst because of lacking MPO in cytoplasm.

The immune response of Japanese flounder (Paralichthys olivaceus) against Neobenedenia girellae (Hargis, 1955) (Monogenea : Capsalidae) was investigated. An acquired protection of fish against secondary infection with the parasite was demonstrated by a reduction in the number and body size of parasites on primed fish which had previously been infected with N. girellae and treated by freshwater bath. In the same experiment, however, no raise in the level of antibody in the sera of primed fish was detected by the enzyme-linked immunosorbent assay (ELISA). In another experiment, although antibody was detected by ELISA in the sera of fish immunized by injection with sonicated parasite antigen, there was no significant difference in the parasite counts between antigen-injected and PBS-injected control fish when challenged with oncomiracidia of N. girellae. These results indicate that the protection induced by the previous infection was not associated with the humoral antibody.

The cell-free extracellular products of a reference isolate of Flexibacter psychrophilus were analyzed by substrate gel electrophoresis (SDS-PAGE), and proteases with apparent molecular masses (AMM) of 114 and 152 kiloDaltons (kDa), which had activity against both casein and gelatin, were identified. Proteases with AMMs from 32 to 86 kDa, which were active against gelatin but not casein, were also observed. Analysis of 28 additional isolates of F. psychrophilus indicated that the isolates formed four groups based on the presence or absence of certain proteases visualized by substrate SDS-PAGE. Seven F. psychrophilus isolates from coho salmon Oncorhynchus kisutch and ayu Plecoglossus altivelis reared in Japan were included in the study. These isolates had protease patterns similar to isolates from the USA and could also be grouped. There was a correlation between protease group and the host from which the bacterium was isolated. Of the 29 isolates studied, all 11 group-1 isolates were from coho sa...

On the basis of the 16S rRNA sequence data analysis among the closely related species, the specific primers for Cytophaga psychrophila were constructed. The specificity in amplifying the 16S rRNA of C. psychrophila was confirmed by using some selected strains of the related species. In addition, it was revealed that these specific primers distinguished C. psychrophila from other principal fish pathogens. The present PCR technique is expected to be a powerful tool for the diagnosis of cold-water disease.

The serological characteristics of the strains of Cytophaga psychrophila isolated from different fishes in different areas were compared by the microtiter method. Included were 3 strains from coho salmon (Oncorhynchus kisutch) in Miyagi Prefecture, 4 from rainbow trout (Oncorhynchus mykiss) in Nagano and Tokyo Pref., 6 from ayu (Plecoglossus altivelis) in Tokushima and Wakayama Pref. In addition to these Japanese strains, 3 strains from coho salmon in USA were used as references. Three antisera were prepared with the type strain NCMB1947 from coho salmon in USA, FPC828 from coho salmon in Miyagi, and FPC840 from ayu in Tokushima. In the comparison of C. psychrophila strains by using these antisera, all were positively agglutinated by each of the antisera, indicating the presence of common antigen (s). On the basis of the absorption analysis with thermostable antigens, two distinct serotypes (O-1 and O-2) were recognized. The strains from coho salmon in Japan and USA belonged to serotype O-1. Serotype O-2 occurred only in Japan, and was distributed among rainbow trout as well as ayu.

A field survey indicated that Branchiura sowerbyi was the only species of oligochaete in a KED (kidney enlargement disease) -enzootic goldfish farm and that the oligochaete was infected with four species of actinosporeans : Neoactinomyxon sp., Raabeia sp. and two species of Aurantiactinomyxon. When goldfish showing external symptoms of KED were kept together with B. sowerbyi for 2-7 months, the prevalence of infection of B. sowerbyi with Neoactinomyxon sp. significantly increased. It was thus suggested that the myxosporean Hoferellus carassii transformed into the actinosporean Neoactinomyxon sp. in the oligochaete B. sowerbyi. Specific pathogen free goldfish became infected with H. carassii after coexistence with the oligochaete releasing Neoactinomyxon sp. for 4 months.

Phagocytic activity of the pronephric and peripheral blood neutrophils was studied in the eel, Anguilla japonica, injected with physiological saline or bacterial cells. In phagocytosis, i.e. phagocytic rate and phagocytic index, and chemiluminescent response, the neutrophils from the bacteriainjected eel showed higher activity than those from the saline-injected eel, and the peripheral blood neutrophils were more active than the pronephric ones. It is concluded that in response to the bacterial stimulus, the hematopoietic tissues of the kidney supply neutrophils with higher phagocytic activity to the peripheral blood.

Columnaris disease, Flexibacter columnaris infection, was first described in North America. Recent publications indicate that its distribution is worldwide. Both warmwater and coldwater fish are infected with F. columnaris. All freshwater fish are probably susceptible to the disease under environmental conditions favourable to the bacterium and stressful to the fish. Flexibacter columnaris cannot tolerate sea water. Generally, outbreaks occur when the average water temperatures reach 15°C. The bacterium apparently enters the fish through the skin and gills. The swarms of bacteria involved give the lesions a characteristic yellow pigmentation. The external lesions destroy the skin, muscle and gill tissues. It is relatively easy to isolate the bacteria from the internal organs of the diseased fish, but there is no evidence of internal tissue damage. In this short review, emphasis is placed on several factors relative to water environment which play a role in the development of outbreaks of columnaris disease: 1 The optimum temperature for an outbreak of F. columnaris infection is thought to be between 20 and 30°C. Mortalities seldom occur at temperatures below 15°C, although highly virulent strains can kill fish at lower temperatures. 2 Flexibacter columnaris can survive longer in water of normal to high hardness, as well as in water containing small amounts of sodium, potassium, calcium or magnesium salts, but it cannot live in sea water. 3 Flexibacter columnaris grows well on paniculate fish feeds so that such deposits in the water may act as reservoirs of infection. 4 An important source of infection is feral fish. In the Columbia River or the Fraser River, resident coarse fish such as suckers, Catostomus spp., are thought to transmit the infection to returning adult salmon. 5 Flexibacter columnaris fails to invade fish in the presence of competitive bacteria like Citrobacter freundii when its initial numbers are approximately one in a 100 less than those of the competitors. 6 Crowding seems to be not only a stressor for fish, but also a factor that increases the chance of adhesion of F. columnaris to the external tissues of fish.

Infectivity of Flexibator columnaris to loach, Misgurnus anguillicaudatus (Blecker, Gunther), was studied with variation conditions and the presence of other bacteria in the water medium. Invasion of fish by the poathogen varied with different water conditions. The highest prevalence of infection was observed in an experimentally formulated water containing 0·03% NaCl 0·01% KCl, 0·002% CaCl22H2O and 0·004% MgCI2 6H2O. The percentage of infection in tap water was similar to that of the formulated water. The infeetion rate was low in the individual salt solutions, and no infection occurred in distilled water. Even in the formulated water, the pathogen failed to infect the fish when Citrobacter freundii, Aeromonas hydrophila or Pseudomonas fluorescens were present at densities 100 times that of F. columnaris. However, in such cases, the numbers of F. columnaris in the mucus of the fish body surface did not increase much throughout the experimental period. In contrast, F. columnaris suceessfully invaded the fish in the presence of either Streptococcus sp., Flavobacterium flavescens or Flavobacterium fuscum, even when the numbers of these bacteria were 1000 times that of F. columnaris. In this ease, F. columnaris gradually increased its numbers on the fish body surface.