Kazuma Yoshikoshi

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.

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.

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...

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 ...

Structural characterization of Tn916-like element in Streptococcus parauberis serotype II strains isolated from diseased Japanese flounder

Aims:  To screen for the existence and determine the structure of Tn916‐like element in Streptococcus parauberis serotype II strains isolated from cultured Japanese flounder in western Japan.

Effects of Exogenous Lipid Peroxides on Mortality and Tissue Alterations in Japanese Pearl Oysters Pinctada fucata martensii

Abstract The effects of exogenous lipid peroxides, suspected to be a cellular injury factor that causes mass mortalities of cultured Japanese pearl oysters Pinctada fucata martensii, were investigated in vivo and in vitro. Cumulative mortalities of experimental oysters exposed to oxidized oils in suspension (fish feed oil) or emulsion (methyl linoleate) were approximately 40% after 8 or 9 weeks, whereas mortalities were 0.0% (suspension) and 5.6% (emulsion) in control oysters exposed to unoxidized oils. Pathological changes observed in experimental oysters were characterized by blebbing and necrosis of cells in various organs that had spread from the digestive organ and were identical to those observed in diseased oysters from natural mass mortalities. The thiobarbituric acid values of experimental oysters were consistently and significantly higher than those of control oysters. An in vitro exposure examination also demonstrated that oxidized oil caused conspicuous blebbing and necrosis in the epithelial ...

An Investigation of the Pathology Associated with Mass Mortality Events in the Cultured Japanese Pearl Oyster Pinctada fucata martensii at Four Farms in Western Japan

Abstract The epidemiological and histopathological characteristics of the mass mortality of cultured Japanese pearl oysters Pinctada fucata martensii were investigated. Rearing experiments with Japanese pearl oysters in farms revealed that the mass mortality occurs as a regular annual event in particular farms in western Japan. Diseased oysters had marked atrophy and red-brown discoloration of the soft parts of the body. Light microscopy revealed that the epithelia of the stomach, the ducts of the digestive diverticula (DD), and the DD themselves showed marked blebbing and necrosis to varying degrees during earlier stages of the disease. At advanced stages, muscle fibers of the adductor muscle, heart, mantle, and other parts of the body and the connective tissues of various organs involving the vascular system also exhibited considerable atrophy and necrosis. There were no remarkable changes in the branchial and pallial epithelia. No viral, bacterial, mycotic, or parasitic causative organisms were found i...