UNLABELLED: With the rapid growth of inland aquaculture worldwide, side effects such as the discharge of nutrients and antibiotics pose a threat to the global environments. A sustainable future for aquaculture requires an effective management system, including the early detection of disease through the monitoring of specific biomarkers in aquaculture tanks. To this end, we investigated whether fish feces in aquaculture tanks could be used for non-invasive health monitoring using ayu (Plecoglossus altivelis) infected with Flavobacterium psychrophilum, which causes bacterial cold-water disease worldwide. Feces that were subsequently produced in the tanks were used for metagenomic and metabolomic analyses. The relative abundances of the genera Cypionkella (0.6% ± 1.0%, 0.1% ± 0.2%), Klebsiella (11.2% ± 10.0%, 6.2% ± 5.9%), and F. psychrophilum (0.5% ± 1.0%, 0.0% ± 0.0%) were significantly higher in the feces of the infection challenge test tanks than in those of the control tanks. The abundances of cortisol, glucose, and acetate in the feces of the infection challenge test tanks were 2.4, 2.4, and 1.3 times higher, respectively, than those of the control tanks. Metagenome analysis suggested that acetate was produced by microbes such as Cypionkella. The abundances of indicated microbes or metabolites increased after day 4 of infection at the earliest, and were thus considered possible biomarkers. Our results suggest that feces produced in aquaculture tanks can potentially be used for non-invasive and holistic monitoring of fish diseases in aquaculture systems. IMPORTANCE: The aquaculture industry is rapidly growing, yet sustainability remains a challenge. One crucial task is to reduce losses due to diseases. Monitoring fish health and detecting diseases early are key to establishing sustainable aquaculture. Using metagenomic and metabolomic analyses, we found that feces of ayu infected with Flavobacterium psychrophilum contain various specific biomarkers that increased 4 days post-challenge, at the earliest. Our findings are the first step in establishing a novel, non-invasive, and holistic monitoring method for fish diseases in aquaculture systems, especially in ayu, which is an important freshwater fish species in Asia, promoting a sustainable future.

Abstract Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease, is a devastating, worldwide distributed, fish pathogen causing significant economic loss in inland fish farms. Previous epidemiological studies showed that prevalent clonal complexes (CC) differ in fish species affected with disease such as rainbow trout, coho salmon and ayu, indicating significant associations between particular F. psychrophilum genotypes and host species. Yet, whether the population structure is driven by the trade of fish and eggs or by host-specific pathogenicity is uncertain. Notably, all F. psychrophilum isolates retrieved from ayu belong to Type-3 O antigen (O-Ag) whereas only very few strains retrieved from other fish species possess this O-Ag, suggesting a role in outbreaks affecting ayu. Thus, we investigated the links between genotype and pathogenicity by conducting comparative bath infection challenges in two fish hosts, ayu and rainbow trout, for a collection of isolates representing different MLST genotypes and O-Ag. Highly virulent strains in one host species exhibited low to no virulence in the other. F. psychrophilum strains associated with ayu and possessing Type-3 O-Ag demonstrated significant variability in pathogenicity in ayu, ranging from avirulent to highly virulent. Strikingly, F. psychrophilum strains retrieved from rainbow trout and possessing the Type-3 O-Ag were virulent for rainbow trout but not for ayu, indicating that Type-3 O-Ag alone is not sufficient for pathogenicity in ayu, nor does it prevent pathogenicity in rainbow trout. This study revealed that the association between a particular CC and host species partly depends on the pathogen’s adaptation to specific host species.

“Funazushi” is a traditional fermented fish product in Japan. The bacterial flora and aroma compounds of funazushi were investigated to elucidate the manufacturing method preferred by modern people. Funazushi was classified into 6 groups by dominant bacteria such as Lactobacillus spp., Lb. acidipiscis, Lb. buchneri, Lb. brevis, Bacillus spp. and Staphylococcus spp.. The characteristics of the aroma components detected in the funazushi could be divided into three groups, corresponding to the top, middle, and bottom funazushi in the competition score, respectively. Among the funazushi samples that received high scores in the competition, many aroma compounds common to sake were detected. On the other hand, rancid odor components were detected in the low scoring samples. It was shown that the preference for funazushi can be explained by the aroma compounds. On the other hand, no clear relationship between preference and bacterial flora was observed. Temporal trend of the variation on bacterial flora during fermented period was required.

Flavobacterium psychrophilum can infect rainbow trout Oncorhynchus mykiss and ayu Plecoglossus altivelis, detrimentally affecting global aquaculture. ​This study aimed to understand the infectivity and host specificity of two F. psychrophilum strains, SG950607 and PH0424, which were isolated from the kidneys of rainbow trout and ayu, respectively. ​Rainbow trout (fry) and ayu (adult and fry) were infected via two methods: bath immersion and intraperitoneal injection. ​We investigated the ability of each F. psychrophilum strain to survive in the serum of non-original host fish species. ​Rainbow trout died upon being infected by PH0424. ​In addition, the complement of the rainbow trout did not show a bactericidal effect against PH0424. ​On the other hand, ayu infected with SG950607 survived, and their complement eliminated this strain. ​The difference in mortality rates is likely associated with the susceptibility of each strain to the complement activity possessed by the host fish.

Rotifer production without contamination by undesirable organisms is important for obtaining high yields of healthy fish. In this study, we investigated the suitability of the biodegradable polymer poly(butylene succinate-co-adipate) (PBSA) for rotifer production, focusing on the effects of PBSA addition on the growth of rotifers (Brachionus plicatilis), Euplotes ciliates, and Vibrio bacteria. PBSA addition enhanced rotifer growth in the tested range of 0.5-50 mg/ml. This effect was dose-dependent; the higher the concentration, the greater the effect. The maximum intrinsic and daily growth rates were 0.519 and 68%, respectively, equivalent to or higher than those under optimal rotifer culture conditions. In contrast, inhibitory effects on Euplotes growth were observed at 1 mg/ml or higher concentrations of PBSA, in a dose-dependent manner. Though it remains unclear if PBSA directly inhibits the growth of Vibrio bacteria, culture water of well-grown rotifers had fewer Vibrio bacteria. PBSA addition demonstrated pH-decreasing and buffering functions; the pH value in rotifer-free culture water decreased from 7.9 to 6.7 after a 5-day incubation period and to 5.8 after a 10-day incubation period. This study demonstrates that the application of PBSA in aquaculture could enhance rotifer production while avoiding contamination by Euplotes and Vibrio.

Rainbow trout fry syndrome (RTFS) and bacterial coldwater disease (BCWD) is a globally distributed freshwater fish disease caused by Flavobacterium psychrophilum. In spite of its importance, an effective vaccine is not still available. Manipulation of the microbiome of skin, which is a primary infection gate for pathogens, could be a novel countermeasure. For example, increasing the abundance of specific antagonistic bacteria against pathogens in fish skin might be effective to prevent fish disease. Here, we combined cultivation with 16S rRNA gene amplicon sequencing to obtain insight into the skin microbiome of the rainbow trout (Oncorhynchus mykiss) and searched for skin bacteria antagonistic to F. psychrophilum. By using multiple culture media, we obtained 174 isolates spanning 18 genera. Among them, Bosea sp. OX14 and Flavobacterium sp. GL7 respectively inhibited the growth of F. psychrophilum KU190628-78 and NCIMB 1947(T), and produced antagonistic compounds of<3 kDa in size. Sequences related to our isolates comprised 4.95% of skin microbial communities, and those related to strains OX14 and GL7 respectively comprised 1.60% and 0.17% of the skin microbiome. Comparisons with previously published microbiome data detected sequences related to strains OX14 and GL7 in skin of other rainbow trout and Atlantic salmon.

We collected 28 and 10 Flavobacterium psychrophilum strains isolated from dead and diseased ayu Plecoglossus altivelis altivelis, respectively, from several rivers of Kochi Prefecture. A total of 38 strains were identified from 10 gene regions by using the on/off switch assay, polymerase chain reaction-restriction fragment length polymorphism, and multilocus sequence typing. Genotypes of F. psychrophilum isolated from dead ayu were mostly G-C/A/S/52 (22/28) whereas genotypes of F. psychrophilum isolated from diseased ayu were G-C/A/S/52 (6/10) and A-C/A/R/45 (4/10). The number of outbreaks of bacterial cold-water disease (BCWD) caused by F. psychrophilum in ayu in the rivers of Kochi Prefecture increased in 2015 and peaked in 2018. Most F. psychrophilum isolates from dead ayu in 2015-2018 were identified as having genotypes of G-C/A/S/52 (22/24). These results suggest that the outbreak of BCWD in ayu in the rivers of Kochi Prefecture was caused by a genotype of G-C/A/S/52.

Flavobacterium psychrophilum can be divided into three genotypes, G-C type (ayu type), A-C type (multi-fish type), and A-T type (salmon-trout type), by two single-nucleotide polymorphisms in the DNA gyrase subunit A (gyrA) gene. We isolated F. psychrophilum from various samples collected from the lower basin of a river flowing into Lake Biwa, as a model for bacterial survey, in Shiga Prefecture in June, September, and December from 2010 to 2013 and investigated their gyrA genotypes. All three types of F. psychrophilum were isolated in June when ayu went up the river from the lake. In September, ayu gathered in the lower river basin for spawning and became high in density, almost all of the isolates were of the G-C genotype and the A-T genotype was never isolated. In December, only the A-T type was isolated from the river samples, when Biwa trout Oncorhynchus masou rhodurus came to the river to spawn and were present in the sampling area. In accordance with the seasonal change of the host fish species in the river, the genotype of F. psychrophilum isolated from the environment seems to have changed as well.

Flavobacterium psychrophilum, the etiological agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish, is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide. In this study, the genomic diversity of the F. psychrophilum species is analyzed using a set of 41 genomes, including 30 newly sequenced isolates. These were selected on the basis of available MLST data with the two-fold objective of maximizing the coverage of the species diversity and of allowing a focus on the main clonal complex (CC-ST10) infecting farmed rainbow trout (Oncorhynchus mykiss) worldwide. The results reveal a bacterial species harboring a limited genomic diversity both in terms of nucleotide diversity, with ~0.3% nucleotide divergence inside CDSs in pairwise genome comparisons, and in terms of gene repertoire, with the core genome accounting for ~80% of the genes in each genome. The pan-genome seems nevertheless "open" according to the scaling exponent of a power-law fitted on the rate of new gene discovery when genomes are added one-by-one. Recombination is a key component of the evolutionary process of the species as seen in the high level of apparent homoplasy in the core genome. Using a Hidden Markov Model to delineate recombination tracts in pairs of closely related genomes, the average recombination tract length was estimated to ~4.0 Kbp and the typical ratio of the contributions of recombination and mutations to nucleotide-level differentiation (r/m) was estimated to ~13. Within CC-ST10, evolutionary distances computed on non-recombined regions and comparisons between 22 isolates sampled up to 27 years apart suggest a most recent common ancestor in the second half of the nineteenth century in North America with subsequent diversification and transmission of this clonal complex coinciding with the worldwide expansion of rainbow trout farming. With the goal to promote the development of tools for the genetic manipulation of F. psychrophilum, a particular attention was also paid to plasmids. Their extraction and sequencing to completion revealed plasmid diversity that remained hidden to classical plasmid profiling due to size similarities.

Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997) for a set of 34 strains, we identified key molecular determinants of the serotypes. This knowledge allowed us to develop a robust multiplex PCR-based serotyping scheme, which was applied to 244 bacterial isolates. The results revealed a striking association between PCR-serotype and fish host species and illustrate the use of this approach as a simple and cost-effective method for the determination of F. psychrophilum serogroups. PCR-based serotyping could be a useful tool in a range of applications such as disease surveillance, selection of salmonids for bacterial coldwater disease resistance and future vaccine formulation.

Phosphorothioate modification of DNA and the corresponding DNA degradation (Dnd) phenotype that occurs during gel electrophoresis are caused by dnd genes. Although widely distributed among Bacteria and Archaea, dnd genes have been found in only very few, taxonomically unrelated, bacterial species so far. Here, we report the presence of dnd genes and their associated Dnd phenotype in two Flavobacterium species. Comparison with dnd gene clusters previously described led us to report a noncanonical genetic organization and to identify a gene likely encoding a hybrid DndE protein. Hence, we showed that dnd genes are also present in members of the family Flavobacteriaceae, a bacterial group occurring in a variety of habitats with an interesting diversity of lifestyle. Two main types of genomic organization of dnd loci were uncovered probably denoting their spreading in the phylum Bacteroidetes via distinct genetic transfer events. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

The bacterium Flavobacterium psychrophilum is a serious problem for salmonid farming worldwide. This study investigates by multilocus sequence typing (MLST) the population structure of this pathogen in Japan where it is also a major concern for ayu, a popular game fish related to salmoniforms. A total of 34 isolates collected across the country and 80 isolates sampled in a single model river by electrofishing were genotyped. The data accounting for 15 fish species allowed identifying 35 distinct sequence types (ST) in Japan. These ST are distinct from those reported elsewhere, except for some ST found in rainbow trout and coho salmon, two fish that have been the subject of intensive international trade. The pattern of polymorphism is, however, strikingly similar across geographical scales (model river, Japan, world) in terms of the fraction of molecular variance linked to the fish host (∼50%) and of pairwise nucleotide diversity between ST (∼5 Kbp§ssup§-1§esup§). These observations go against the hypothesis of a recent introduction of F. psychrophilum in Japan. Two findings were made that are important for disease control: 1) at least two independent F. psychrophilum lineages infect ayu and 2) co-infections of the same individual fish by different strains occur. © 2013 Fujiwara-Nagata et al. licensee BioMed Central Ltd.

We developed a simple genotyping method for Flavobacterium psychrophilum for analysing two single nucleotide polymorphisms (SNPs) in the gyrA gene and to distinguish between isolates that are virulent and avirulent to ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel). The genotyping method is an on/off switch assay and is based on the polymerase chain reaction technique with phosphorothioated primers. We classified 232 isolates from four families of fish (i.e. Plecoglossidae, Osmeridae, Cyprinidae and Salmonidae) into four genotypes (G-C, A-T, A-C and G-T). The G-C type isolates exhibited strong pathogenicity to ayu, whereas the A-T and G-T types did not show any pathogenicity to this species. The A-C type exhibited no or weak pathogenicity to ayu. These results indicate that genotyping F.similar to psychrophilum isolates with two SNPs from gyrA can clearly distinguish between isolates potentially harmful to ayu (G-C type) and those that are potentially not harmful or less harmful (A-C, A-T and G-T type). The on/off switch assay provides a quick, simple, and very powerful DNA genotyping technique for F.similar to psychrophilum isolates.

Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum leads to heavy mortality of ayu Plecoglossus altivelis in Japan. Previously, a 28 degrees C warmed water treatment was shown to be an effective treatment for BCWD, since F psychrophilum can not grow and maintain colonies at this temperature. However, it was unclear whether the bacteria might resuscitate after the treatment was over. Therefore, we investigated colony formation, membrane potential and pathogenicity of F psychrophilum at various temperatures (15 degrees C, 23 degrees C, 28 degrees C and 33 degrees C) in sterilized underground water. Within 2 days at 28 degrees C, F psychrophilum completely lost their colony-forming abilities but still maintained their membrane potentials. It seemed that these cells entered into viable but non-culturable (VBNC) state. However, experimental ayu infection revealed that VBNC F psychrophilum cells were unable to cause BCWD, suggesting that the cells were progressing towards death at 28 degrees C.

Bacterial cold-water disease (BCWD) causes heavy mortality of ayu Plecoglossus altivelis in Japan. To control BCWD of ayu, warmed water treatment at 28 degrees C has been used. However, the mechanism of this treatment is unknown. Hence, we investigated the growth/survival of Flavobacterium psychrophilum at 15-28 degrees C under two nutritional conditions: in modified cytophaga (MCY) broth and sterilized underground water. Within 2 days at 28 C in both the conditions, F psychrophilum totally lost its colony-forming ability. We also studied distributions of F psychrophilum in infected fish organs and in rearing water after various treatments. For bacteria detection, the colony-counting method and loop-mediated isothermal amplification (LAMP) method were used. For warmed water treatment, the rearing water was warmed from 18 degrees C to 23 degrees C or 28 degrees C for 3 days. The treatments were started 1 day after immersion infection. In the experimentally infected fish, F psychrophilum was detected neither in any fish organs nor in the rearing water after the 28 degrees C treatment for 3 days. These results indicate the effectiveness of the warmed water treatment at 28 degrees C against BCWD of ayu.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout fry syndrome of salmonids. The pathogen has been reported from all regions in the world involved in salmonid aquaculture, but also from natural fresh-water environments. We established a quantitative loop-mediated isothermal amplification of DNA (LAMP) method to estimate quantities of F. psychrophilum. LAMP primers were designed based on the sequence of the DNA topoisomerase IV subunit B gene, parE, of F. psychrophilum. parE LAMP exhibited a high specificity for the parE gene of F. psychrophilum but not for other related species. parE LAMP detected the gene in a wide range of concentrations from 2.0 x 101 to 2.0 x 109 copies/reaction within 70 min and revealed a good correlation between threshold times and gene copy number.

Vibrio anguillarum kills various kinds of fish over a range of salinities from sea water to fresh water, and causes serious damage to aquaculture systems. In this study, the transcriptional regulation of the Na+-NADH:quinone oxidoreductase (Na+-NQR) operon in V. anguillarum from the logarithmic to stationary phases was investigated. Cloning of the Na+-NQR operon revealed a 7 kb nucleotide sequence composed of six open reading frames with amino acid sequence identity of more than 80% with other Vibrio species. Two promoters, nqrP1 and nqrP2, were identified in the region upstream of the nqrA gene using an S1 nuclease assay. The nqrP1 promoter was constitutively activated throughout the logarithmic to stationary phases and possessed -10 (5'-TAGACT-3') and -35 (5'-ATGGCA-3') sequences, which were similar to the consensus sequence of Escherichia coli. On the other hand, the nqrP2 promoter was activated only at the stationary phase and had only a -10 (5'-CATACT-3') and not a -35 sequence. These results suggest that nqrP2, which works specifically in the stationary phase, contributes to starvation-survival in V. anguillarum.

環境水におけるアユ冷水病菌Flavobacterium psychrophilumの定量的モニタリング．

Vibrio anguillarum kills various kinds of fish over salinities ranging from seawater to freshwater. In this study, we investigated the role of Na+ in V. anguillarum, especially under energy-depleted conditions such as in natural seawater. V. angustum S14, which is a typical marine vibrio. was used for comparison. V. anguillarum only required Na+ for starvation-survival, but ill contrast, V. angustum S14 always required Na+ for both growth and starvation-survival. In marine vibrios, Na+ is used in the Na+-dependent respiratory chain that produces the sodium motive force (SMF) across the cell membrane. It has been considered that marine vibrios always need a SMF produced by Na+, however ill the case of V. anguillarum, the SMF is not required for growth, but becomes more important for starvation-survival. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

The activity of membrane-bound NADH oxidase of Vibrio anguillarum M93 (serotype J-O-1), which causes vibriosis in freshwater area was activated by Na+ in the same manner as other marine Vibrios. However, in addition to Na+, K+ was also found to positively enhance the NADH oxidase activity of strain M93. This tendency has not been recognized in other marine Vibrios. Furthermore, the Na+-dependent NADH oxidase of strain M93 required less Na+ (0.1 M) for its maximum activity than those of other Vibrios such as Vibrio alginolyticus and 'Vibrio angustum' S14, which were in the range of 0.4 M NaCl, similar as seawater. Destruction of H+ motive force by a proton conductor carbonylcyanide m-chlorophenylhydrazone (CCCP) revealed high dependency of V anguillarum on the primary H+ pump. Even at pH 8.5, V anguillarum strains other than serotype O-4 could not grow well with the addition of CCCP. In contrast, marine-type Vibrios such as V. alginolyticus and V. angustum S14 can grow well at pH 8.5 even with the addition of CCCP. The lower requirement for Na+ in V anguillarum probably reflects the salinity of their original habitats. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Vibriosis caused by Vibrio anguillarum seriously injures freshwater fish (Salmoniforms) almost every year in Lake Biwa, Japan. This pathogen needs NaCl for its growth and survival. When the pathogen was directly exposed to sterilized aged lake water (ALW) at room temperature, it suddenly lost its culturability and pathogenicity, and died within half a day due to the low osmolarity. In this report, the survival of the pathogen as biofilms formed on air-solid and liquid-solid (agar or polystyrene) interfaces in ALW was investigated. When the biofilms formed at air-solid and liquid-solid (agar or polystyrene) interfaces were exposed to ALW at 4-5°C in the dark, the pathogen survived for more than 2 and 4 weeks, respectively. The biofilms at both interfaces at 4-5°C in the dark enhanced the production of a mucous polymer matrix. The main constituent of the polymer was exopolysaccharide. The polymer was produced only in the dark at low temperature. At 20°C, there was no production of the polymer and the survival of the pathogen was shortened. The biofilm seemed to provide a functional consortium to support the survival of V. anguillarum in freshwater. © 2003, Japanese Society of Microbial Ecology & The Japanese Society of Soil Microbiology. All rights reserved.

Vibriosis caused by Vibrio anguillarum serotype J-O-1 seriously affects the freshwater fish ayu (Plecoglossus altivelis) in Lake Biwa, Japan. Survival patterns of V. anguillarum were investigated in aged lake water (ALW) supplemented with or without 0.75% NaCl. It was found that 0.1-1.0% of V. anguillarum cells maintained the ability to form colonies even after 6 weeks in 0.75% NaCl-ALW. Under the same starved conditions, MPN counts with liquid medium were 100 times higher than CFU counts. When exposed to sterilized aged lake water without NaCl, V. anguillarum entered a non-culturable state within half a day. As the mineralization activity of non-culturable cells was still 0.1-5.4% of that in the culturable phase, this physiological state can be described as “viable but non-culturable”. However, all attempts to return to a culturable state including re-infection were un-successful. The non-culturable cells in ALW lost all pathogenicity in fish. Since a longer exposure to ALW resulted in less mineralization activity, the non-culturable state of V. anguillarum cells in freshwater seems to reflect a phase of decay leading to cell death. However, certain environmental factors such as coldness and microaerobiosis seem to help the pathogen to survive longer in freshwater without a loss of culturability. © 2003, Japanese Society of Microbial Ecology & The Japanese Society of Soil Microbiology. All rights reserved.

Vibriosis caused by Vibrio anguillarum seriously damages freshwater fish (Salmoniforms) almost every year in Lake Biwa, Japan. This pathogen requires NaC1 for its growth. When the pathogen was exposed to sterilized aged lake water, it lost its culturability without loosing respiratory activity. Although this physiological state appears to be 'viable but nonculturable' (VBNC), the cells could not be resuscitated even in host fish bodies. As these cells totally lost their pathogenicity, the VBNC-like state is probably on the pathway to cell death. Environmental factors such as darkness, coldness, and anaerobiosis helped the pathogen to survive in freshwater without reaching a nonculturable state.

申請者らは先行研究において、アユ由来冷水病菌が3型O抗原関連遺伝子を持つこと、国内外のニジマス由来冷水病菌の中にはアユ由来冷水病菌と同じ3型O抗原関連遺伝子を持つ株が少数いることを明らかにした。本課題では、3型O抗原関連遺伝子が、アユあるいはニジマスに対する病原性の発揮に重要なのかを解明するために、アユならびにニジマスへの感染試験を行いそれらの病原性の有無を確認した。また、アユ由来で3型以外のO抗原関連遺伝子を持つ冷水病菌も発見されたので、その病原性についても調べた。 供試した冷水病菌は、3型O抗原関連遺伝子を持つ13株（アユ由来：10株、サケ科魚類由来：3株）、0型O抗原関連遺伝子を持つ2株（アユ由来：1株、コイ科魚類由来：1株）、そして2型O抗原関連遺伝子を持つ2株（サケ科魚類由来）の合計17株である。アユ（海産系）の稚魚とニジマスを用いて浸漬感染により攻撃試験を行った。 その結果、3型O抗原関連遺伝子を持つニジマス由来の冷水病菌はニジマスに対してのみ病原性を示し、アユには病原性を示さなかった。3型O抗原関連遺伝子を持つアユ由来の冷水病菌は、1株を除いて全てアユに対してのみ病原性を示し、ニジマスには病原性を示さなかった。0型O抗原関連遺伝子を持つアユ由来の冷水病菌は、ニジマスにもアユにも病原性を示さなかった。0型O抗原関連遺伝子を持つコイ科魚類由来の冷水病菌も同様に、ニジマスにもアユにも病原性を示さなかった。2型O抗原関連遺伝子を持つ2株（サケ科魚類由来）は、アユには病原性を示さなかったが、ニジマスに対しては病原性を示した。

水産養殖魚類の疾病は大きな経済損失である。特に、アユ養殖において深刻な冷水病には有効な予防策がない。そこで、魚類体表面に善玉菌を導入する技術を開発し、アユの病気の予防・治療を目指す。本課題では、投与方法、善玉菌の投与量や組み合わせを検討し、類似技術よりも有効率や汎用性の高い技術を開発し、起業の可能性を検証する。

魚類養殖漁場の有機汚濁の程度は、硫化物の発生・蓄積や底生生物の減少といった汚濁の結果で表現されている。海域が持つ自然の自浄作用に頼った漁場管理の場合、汚濁の結果を見ていては手遅れになってしまう。悪化を予防するためには、悪化が起きる予兆を知る指標 の方が役にたつ。申請者らは硫酸還元菌による硫化水素の生成が有機酸（主に乳酸）濃度によって制御されている点に注目し、底泥の乳酸菌数と魚類養殖による負荷（主に硫化物量）との関係を調べてきた。その結果、生簀下の硫化物量が増える前に乳酸菌数が増えることな どを明らかにした。乳酸菌の変動は、硫化物量の増大する前触れとなっている可能性が高い。しかし、乳酸菌の数や種組成の変化と相関関係を示す硫化物量の水準や、両者の変動にどのくらいのタイムスパンがあるのかを含めて未解明な点が残されている。本研究ではこれらの 解明に取り組み、硫化物量が基準値内であっても進行する汚濁を検知する「隠れメタボ診断」の様なツールの開発を目指す。

Many beautiful corals are living on the ropes of tuna cages in Amami Experimental Station of Kinki University. This suggests that the appropriate aquaculture activities would be done in this area. Such co-existence between aquaculture and coral is very rare in the world. In this study, we focused on the ecological functions of marine bacteria and coral mucus as the first step to understand the wonderful balance of the ecosystem. The mucus was collected from Acropora sp. which was a dominant species on ropes of the tuna cages. Bacterial abundance in the “mucus+seawater” sample during 10-h-incubation increased, while that in the seawater sample (control) was lower and constant. Bacterial composition changed drastically in the “mucus+seawater “ sample, but not in the seawater sample.