Kinya Kanai

Identification of quorum-sensing signal molecules and the LuxRI homologs in fish pathogen Edwardsiella tarda.

Edwardsiella tarda is a gram-negative bacterium that causes septicaemia in fish and serious damage to the aquaculture industry. The virulence factors of this pathogen and control mechanisms of the expression of virulence genes have not yet been clearly elucidated. A number of gram-negative pathogenic bacteria have a quorum-sensing system. These bacteria produce N-acyl-L-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule. In this study, we found that E. tarda isolated from deceased flounder produces AHLs. Thin layer chromatography analysis indicated that the two kinds of AHL produced by E. tarda seemed to be N-hexanoyl-L-homoserine lactone (C6-HSL) and N-heptanoyl-L-homoserine lactone (C7-HSL). We have cloned and sequenced the quorum-sensing genes, luxI homolog (edwI) and luxR homolog (edwR). EdwI and EdwR showed high identity with CarI/CarR and ExpI/ExpR from Erwinia carotovora, respectively. SDS-PAGE analysis of extracellular proteins revealed that the expression of the 55-kDa protein, which was reported as a virulent-strain-specific protein, is controlled by AHLs. These results suggest that some virulence factors are regulated by the quorum-sensing system in E. tarda.

Comparison of the responses of peritoneal macrophages from Japanese flounder (Paralichthys olivaceus) against high virulent and low virulent strains of Edwardsiella tarda

In vivo infection studies in Japanese flounder (Paralichthys olivaceus) demonstrated that the number of viable cells of the virulent strain (NUF251) of Edwardsiella tarda increased gradually in kidney and hepato-pancreas after intraperitoneal injection, but the low virulent strain (NUF194) did not. To gain insight into the virulence factors of E. tarda, in vitro responses of Japanese flounder (P. olivaceus) peritoneal macrophages to these strains were compared in terms of phagocytosis, bactericidal activity, and reactive oxygen species (ROS) generation as measured by chemiluminescence (CL) responses. Microscopic observation revealed that these two strains of E. tarda were phagocytosed by the peritoneal macrophages, and there was no significant difference in the mean numbers of ingested bacteria per macrophage between these strains. A gradual increase in the number of viable cells of the highly virulent strain within macrophages was observed during 9h post-phagocytosis, whereas no significant replication of the low virulent strain within macrophages was detected. These results suggest that the virulent strain of E. tarda has an ability to survive and replicate within macrophages, while the low virulent strain has no such ability. When the peritoneal macrophages were exposed to the opsonized low virulent E. tarda strain, a rapid increase in CL response was induced. However, the highly virulent strain caused only background level of CL response. By the subsequent stimulation with phorbol myristate acetate, the macrophages exposed to the virulent E. tarda strain showed extremely higher CL response than that of the one exposed to the low virulent E. tarda strain. These results suggest that the virulent E. tarda prevents the activation of ROS generation system during phagocytosis, though the system is still capable of responding to other stimulation. The virulent strain significantly reduced the CL response induced by xanthine/xanthine oxidase system, while the low virulent strain had almost no effect. Furthermore, the virulent strain showed greater resistance to H(2)O(2) than the low virulent strain. Our results suggest that the virulent strain of E. tarda is highly resistant to ROS, and such ability might allow the organism to survive and multiply within phagocytes, and may serve to disseminate E. tarda throughout the host during in vivo infection.

Ecological investigation of Streptococcus iniae in cultured Japanese flounder (Paralichthys olivaceus) using selective isolation procedures

Abstract Streptococcus iniae is an important bacterial pathogen of Japanese flounder ( Paralichthys olivaceus ). We investigated the prevalence of S. iniae in Japanese flounder and its cultural environment for two consecutive years in a flounder farm. Direct plating procedure using thallium acetate-oxolinic acid-blood (TAOAB) agar and enrichment culture in a selective broth (SB) followed by subculture on TAOAB agar were employed for detecting S. iniae from samples. Todd–Hewitt broth (1 l) containing 0.5 g thallium acetate, 10 mg colistin sulphate (CS), and 5 mg oxolinic acid (OA) was chosen as a selective broth. On most sampling dates except for those of low water temperature, S. iniae was detected from sediment and water samples of 1-year-old fish tanks. Detection rates of S. iniae for sediment and water samples from 0-year-old fish tanks were low from January, when rearing of fingerling was started in this farm, until the epizootic broke out. Detection rates of S. iniae for samples from 1-year-old fish tanks were always higher than those of 0-year-old fish tanks. S. iniae was detected also from water supply in high temperature months. A total of 15 out of 230 flounder sampled were S. iniae -infected as the pathogen was isolated from their brain and/or kidney. S. iniae was detected frequently from the gill and skin mucus of apparently healthy flounder. Broth enrichment procedure was suited for detection of S. iniae from samples of fish tissue and water supply. Direct plating procedure was suited for sediment samples. For water samples, both procedures should be employed.

Comparative analysis of the production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) from macrophages exposed to high virulent and low virulent strains of Edwardsiella tarda

We previously reported that high virulent strain (NUF251) of Edwardsiella tarda has an ability to prevent the production of reactive oxygen species by macrophages, and is even capable of surviving and multiplying within Japanese flounder (Paralichthys olivaceus) peritoneal macrophages, whereas the low virulent strain (NUF194) has no such ability. In this study, we found that NUF251 and NUF194 induced NO and TNF-alpha production from Japanese flounder peritoneal macrophages, and NUF251 caused faster induction of NO release and much higher level of TNF-alpha production than NUF194. In addition, similar differences between two strains in terms of the induction of NO and TNF-alpha production were also observed in mouse macrophage cell line RAW264.7 cells. Our results suggest that the potent ability to induce the production of NO and TNF-alpha from macrophages may be one of the factors responsible for the virulence of E. tarda.

Identification of a 19.3-kDa protein in MRHA-positive Edwardsiella tarda: putative fimbrial major subunit

The hemagglutinating properties of Edwardsiella tarda isolated from fish were investigated. Hemagglutination of E. tarda was not inhibited by D-mannose but was strongly inhibited by fetuin and N-acetylneuraminic acid. Extraction of hemagglutinating activity from bacterial cells was achieved using n-octyl-beta-D-thioglucoside (NOTG), and the NOTG extracts were fractionated by sucrose density gradient ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the fractions revealed that a 19.3-kDa protein band appeared in the fractions exhibiting highest hemagglutinating activity. In an immunoblot analysis of NOTG extracts from 18 strains of E. tarda, the 19.3-kDa protein was detected only in the extracts possessing hemagglutinating activity. The predicted amino acid sequence of a 534-bp gene encoding the 19.3-kDa protein was identical to fimbrial subunit (FimA) of E. tarda by FASTA homology search. These findings suggest that fimbriae are implicated in the hemagglutination of E. tarda.

Larval stages of the bluefin tuna blood fluke Cardicola opisthorchis (Trematoda: Aporocotylidae) found from Terebella sp. (Polychaeta: Terebellidae).

We found aporocotylid larval stages (sporocysts and cercariae) from five individuals of terebellid polychaete Terebella sp., which were collected from seabed substrate and ropes and floats attached to tuna cages in a tuna farm on the coast of Tsushima Island, Nagasaki, Japan. Nucleotide sequences of the regions of internal transcribed spacer 2 ribosomal DNA and 28S ribosomal DNA from these larval stages were 100% identical to those of Cardicola opisthorchis registered in GenBank. C. opisthorchis is a pathogen causing blood fluke infection of Pacific bluefin tuna Thunnus orientalis, which is considered to have a significant impact on the Japanese Pacific bluefin tuna aquaculture industry. This is the first description of the intermediate host of C. opisthorchis. This indicates that the life cycle of C. opisthorchis is completed within tuna farms in this area.

Histopathological alterations in the digestive system of larval and juvenile Japanese flounder Paralichthys olivaceus reared on four feeding levels.

Abstract The pathological effects of different feeding levels on the organ systems of larval and juvenile Japanese flounder Paralichthys olivaceus (also known as olive flounder) were studied sequentially from 3 to 45 d posthatch. The flounder were fed enriched live foods—the rotifer Brachionus plicatilis and brine shrimp in the genus Artemia–at four feeding levels (L1–L4). The respective food densities at L1, L3, and L4 were 0.5, 2.5, and 5.0 times that at L2. Level L2 is the one conventionally adopted by hatcheries. At that level, the initial density of rotifers is 0.5 individuals/mL of culture water; the density gradually increases to approximately 3.5 individuals/mL after metamorphosis. The initial density of brine shrimp at L2 is 0.1 individuals/mL, which gradually increases to 3 individuals/mL. Light and electron microscopy showed no evidence of viral, bacterial, parasitic, or mycotic invasions in developing Japanese flounder. However, pathological lesions were observed (including adhesions among the...

Extracellular products from virulent strain of Edwardsiella tarda stimulate mouse macrophages (RAW264.7) to produce nitric oxide (NO) and tumor necrosis factor (TNF)-α

We have previously found that high virulent strain (NUF251) of Edwardsiella tarda, but not low virulent strain (NUF194), was able to survive and multiply within Japanese flounder (Paralichthys olivaceus) peritoneal macrophages. Further studies demonstrated that NUF251 induced much higher levels of NO and TNF-alpha productions than NUF194 in both Japanese flounder peritoneal macrophages and mouse macrophage cell line RAW264.7. In this study, we examined the effects of extracellular products (ECP) from two strains of E. tarda on RAW264.7 cells in terms of the induction of NO and TNF-alpha. ECP from NUF251 stimulated RAW264.7 cells to induce NO production in a concentration-dependent manner. The activity of NUF251-ECP completely disappeared by heat-treatment (at 100 degrees C for 5 min), but it could not be removed by dialysis. Polymyxin B, an endotoxin inhibitor, had no effect on NUF251-ECP-induced NO production. These results suggest that active agents in NUF251-ECP responsible for NO induction may be heat-labile high molecular weight substances rather than the cell wall derived endotoxin like substances. Since NO synthase (NOS) inhibitor, l-NAME, suppressed NUF251-ECP-induced NO production, inducible NO synthase (iNOS) in RAW264.7 cells may be a main source of NO. Furthermore, NUF251-ECP-induced high level of TNF-alpha secretion from RAW264.7 cells. Both NO and TNF-alpha productions induced by NUF251-ECP were significantly blocked by a JNK inhibitor. In contrast to NUF251-ECP, no significant activities of NUF194-ECP to induce NO and TNF-alpha productions were detected. SDS-PAGE and subsequent proteomic analysis of ECP from both strains suggested that NUF251-specific protein, which has sequence homology with flagellin, is present in NUF251-ECP as a main component. Our results suggest that the high virulent strain (NUF251) of E. tarda may specifically produce an exotoxin capable of inducing high levels of NO and TNF-alpha from macrophages through the activation of JNK system, and most probable candidate for such exotoxin might be a flagellin-like protein.

Comparative Genomic Characterization of Three Streptococcus parauberis Strains in Fish Pathogen, as Assessed by Wide-Genome Analyses

Streptococcus parauberis, which is the main causative agent of streptococcosis among olive flounder (Paralichthys olivaceus) in northeast Asia, can be distinctly divided into two groups (type I and type II) by an agglutination test. Here, the whole genome sequences of two Japanese strains (KRS-02083 and KRS-02109) were determined and compared with the previously determined genome of a Korean strain (KCTC 11537). The genomes of S. parauberis are intermediate in size and have lower GC contents than those of other streptococci. We annotated 2,236 and 2,048 genes in KRS-02083 and KRS-02109, respectively. Our results revealed that the three S. parauberis strains contain different genomic insertions and deletions. In particular, the genomes of Korean and Japanese strains encode different factors for sugar utilization; the former encodes the phosphotransferase system (PTS) for sorbose, whereas the latter encodes proteins for lactose hydrolysis, respectively. And the KRS-02109 strain, specifically, was the type II strain found to be able to resist phage infection through the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system and which might contribute valuably to serologically distribution. Thus, our genome-wide association study shows that polymorphisms can affect pathogen responses, providing insight into biological/biochemical pathways and phylogenetic diversity.

Effects of Feeding Levels on the Pathological Alterations in the Digestive System and Mortality of Larvae and Juveniles of Pagrus major

Abstract The effects of two feeding levels on the larvae and juveniles of red seabream Pagrus major (also known as madai) were examined beginning with the onset of exogenous feeding until 29 d posthatch. The red seabream were fed enriched live feeds, Brachionus rotundiformis and Artemia spp., at two feeding levels (L1 and L2). At the L1 level, the initial density of the rotifers was 3.0 individuals/mL, and the feeding density was gradually increased to approximately 20.0 individuals/mL up to 29 d posthatch. With Artemia spp., the initial density at L1 was 0.1 individuals/mL (18 d posthatch), increasing gradually up to 1.1 individuals/mL up to 29 d posthatch. The level L1 is the conventional regime adapted by some hatcheries in Japan for red seabream larva and juvenile rearing. The L2 level was the same composition as L1, but the levels of rotifers and Artemia in L2 were five times those of L1. Red seabream larvae and juveniles responded to application of higher feeding levels by ingesting greater amounts ...