Hisatsugu Wakabayashi

Flexibacter maritimus sp. nov., a pathogen of marine fishes.

The name Flexibacter maritimus is proposed for a group of 15 bacterial strains isolated from diseased red sea bream (Pagrus major), black sea bream (Acanthopagrus schlegeli), and rock bream (Oplegnathus fasciatus). These bacteria grew in cytophaga medium prepared with seawater but failed to grow in cytophaga medium supplemented with NaCl. The isolates were gram-negative, flexible rods which exhibited gliding motility on wet surfaces. They did not utilize agar, cellulose, or chitin. The mean guanine-plus-cytosine content of the deoxyribonucleic acids of eight selected strains was 31.8 ± 0.4 mol%. Strain R2 (= NCMB 2514) is designated the type strain of the new species.

Pseudomonas plecoglossicida sp. nov., the causative agent of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis.

A new Pseudomonas species, for which the name Pseudomonas plecoglossicida is proposed, was isolated from cultured ayu (Plecoglossus altivelis) with bacterial haemorrhagic ascites. The causative agent was similar to Pseudomonas putida biovar A in its phenotypic characteristics and on the basis of 16S rRNA gene sequence analysis, but it reduced nitrate to nitrite. Furthermore, it was distinguished phenotypically from Pseudomonas putida biovar A by utilization of D-malate, L-(+)-tartrate, m-tartrate and nicotinate. The levels of DNA-DNA hybridization between the isolate strain FPC 951T and other reference strains of Pseudomonas species, including Pseudomonas putida, were less than 50%. The G+C content of the DNA of FPC 951T was 62.8 mol%. Strain FPC 951T (= ATCC 700383T) is designated the type strain of the new species.

Electrophoretic Detection of Proteases from Selected Strains of Flexibacter psychrophilus and Assessment of Their Variability

Abstract 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...

Neobenedenia girellae (Hargis, 1955) Yamaguti, 1963 (Monogenea: Capsalidae) from cultured marine fishes of Japan.

The monogenean Neobenedenia girellae (Hargis, 1955) Yamaguti, 1963 is redescribed and reported for the first time in Japan. The parasite was recovered from the body surface, fins, and occasionally from the eyes of 14 species, comprising 5 families of cultured marine fishes from several localities in southwestern Japan. Neobenedenia melleni (MacCallum, 1927) sensu Kaneko et al. (1988) from tilapia (Oreochromis mossambicus) in Hawaii is synonymized with this species. Examination of original specimens (syntypes) of N. melleni sensu MacCallum (1927) revealed differences with N. girellae in having a wide and rounded body, a prominently large anterior hamuli, and absence of glands of Goto. This Neobenedenia from Japanese fishes sometimes showed an unusual morphology of the individual parts of the median sclerites. The potential threat of N. girellae to the health of cultured Japanese fishes is indicated by its low host specificity, wide distribution, and ability to cause mortality due to heavy infection. Unregulated importation of amberjack fry (Seriola dumerili) to Japan appears to be the source of N. girellae infection in Japanese fishes since 1991.

Myxobolus cultus n. sp. (Myxosporea: Myxobolidae) in the goldfish Carassius auratus transformed from the actinosporean stage in the oligochaete Branchiura sowerbyi.

A new myxosporean Myxobolus cultus n. sp. was found in experimentally infected goldfish Carassius auratus exposed to actinosporean spores from the oligochaete Branchiura sowerbyi (Tubificidae). At 2-4 mo following initial exposure, spores of M. cultus were observed in the cartilage of goldfish. A lymphocytic infiltrate surrounded the pseudocysts. Some pseudocysts were destroyed and spores had been engulfed by phagocytes. Phagocytized spores were also found in the epidermis of skin and within melanomacrophage centers in the kidney.

ATTACHMENT-INDUCING CAPACITIES OF FISH TISSUE EXTRACTS ON ONCOMIRACIDIA OF NEOBENEDENIA GIRELLAE(MONOGENEA, CAPSALIDAE)

When oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae) were incubated in wells with lyophilized extracts of fish skin epithelia on the bottom, some attached to the well bottom with the haptor unfolded and shed the ciliated epidermal cells. Based on these morphological changes in oncomiracidia, we developed a new assay method to examine the attachment-inducing capacities of various fish extracts for oncomiracidia. Attachment-inducing capacities were found only in extracts of fish skin epithelium and not in other fish extracts. No significant difference in capacities was observed among extracts of skin epithelia of 4 fish species. Wheat germ lectin and concanavalin A suppressed capacities in extracts of skin epithelia of Japanese flounder and yellowtail, respectively. Suppressed capacities were recovered by adding sugars that bound specifically to these lectins. These results indicate that some sugar-related chemical substances that exist specifically in fish epithelium induce the attachment of N. girellae oncomiracidia.

Life Cycle of Hysterothylacium haze (Nematoda: Anisakidae: Raphidascaridinae)

Natural infections with Hysterothylacium haze in the Japanese common goby, Acanthogobius flavimanus, were observed in detail. In gobies in which no worm eggs were deposited, second-stage larvae were found in the digestive tract wall, and third-stage larvae occurred in the digestive tract wall, mesentery, and body cavity, whereas fourth-stage larvae and adults were found in the body cavity. This stage-habitat relationship demonstrates the infectivity of second-stage larvae to the goby and the larval migration. In heavily infected gobies, eggs and all worm stages, from hatched second-stage larvae to adults, often were found together in the body cavity of one individual host, suggesting that hatched second-stage larvae can develop in the body cavity. It was shown experimentally that H. haze develops to the second stage in the egg and does not hatch spontaneously. When a goby was fed the viscera of heavily infected gobies containing eggs and various stages of worms or artificially incubated eggs containing second-stage larvae, second- and third-stage larvae were recovered from the digestive tract wall, and fourth-stage larvae and adults were found in the body cavity. When polychaetes or crustaceans were placed in contact with infected goby viscera or incubated eggs, only second-stage larvae were recovered from the body cavity of the invertebrates. The experimental results were consistent with observations on natural infections and indicate that the direct life cycle of H. haze may involve invertebrates as transport hosts.

Attachment-inducing capacities of fish skin epithelial extracts on oncomiracidia of Benedenia seriolae (Monogenea: Capsalidae).

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.

Respiratory burst assay of head kidney macrophages of ayu, Plecoglossus altivelis, stimulated with Glugea plecoglossi (Protozoa: Microspora) spores

The respiratory burst assay was conducted using the nitroblue tetrazolium reduction method and horseradish peroxidase method to investigate the events when ayu head kidney macrophages phagocytize fresh and formalin-killed Glugea plecoglossi spores. The production of O2 against G. plecoglossi spores was negligible compared to zymosan (P < 0.01). Zymosan-induced O2 production was markedly inhibited by adding G. plecoglossi spores simultaneously (P < 0.01). This phenomenon was dose dependent, and killed spores were less inhibitory than fresh spores. The production of H2O2 was drastically increased when G. plecoglossi spores were added (P < 0.01), and most spores were phagocytized. From these results, it is suggested that G. plecoglossi spores modulate the hosts phagocytic response to establish infection.

Serological Characteristics of Flavobacterium branchiophila Isolated from Gill Diseases of Freshwater Fishes in Japan, USA, and Hungary

Abstract The characteristics of six strains of Flavobacterium branchiophila, isolated from coldwater fishes with bacterial gill disease, were determined and compared by both agglutination and precipitation tests. The bacterial strains used in this study were isolated in Japan (3 isolates), USA (2 isolates), and Hungary (1 isolate). All of the strains were identical in physiological characteristics, except for slight differences in temperature and salinity tolerances. They possessed common antigens; however, the Japanese strains possessed serological differences from both USA and Hungarian strains. The results of the precipitation test identified one antigen specific for Japanese strains and two antigens specific for both USA and Hungarian strains.