Shinpei Wada

Antimycotic Activity of Eugenol against Selected Water Molds

Abstract This study was conducted to investigate the antimycotic activities of eugenol, a major essential oil of clove, against some fish pathogenic species of Saprolegniaceae as well as to determine the toxicity of eugenol to selected cultured fish. Two eugenol solutions were used in this study, a 10% v/v (volume:volume) solution in dimethyl sulfoxide (DMSO) and FA 100, which contains 10% v/v eugenol. The fungal species investigated were Saprolegnia parasitica, S. diclina, S. ferax, S. salmonis, Achlya klebsiana, and Aphanomyces piscicida. The minimum inhibitory concentrations (MICs) of eugenol in DMSO against Saprolegnia spp., A. klebsiana, and A. piscicida were 500, 250, and 125 μg/mL, respectively, and the fungicidal concentrations (for complete killing) were 1,000, 500, and 250 μg/mL, respectively. In contrast, the MICs of FA 100 against S. parasitica, other Saprolegnia spp., A. klebsiana, and A. piscicida were 250, 125, 250, and 63 μg/mL, respectively, and the fungicidal concentrations were 1,000, 1...

Discrimination of Mycobacterium abscessus subsp. massiliense from Mycobacterium abscessus subsp. abscessus in Clinical Isolates by Multiplex PCR

ABSTRACT The rapidly growing mycobacterium M. abscessus sensu lato is the causative agent of emerging pulmonary and skin diseases and of infections following cosmetic surgery and postsurgical procedures. M. abscessus sensu lato can be divided into at least three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Clinical isolates of rapidly growing mycobacteria were previously identified as M. abscessus by DNA-DNA hybridization. More than 30% of these 117 clinical isolates were differentiated as M. abscessus subsp. massiliense using combinations of multilocus genotyping analyses. A much more cost-effective technique to distinguish M. abscessus subsp. massiliense from M. abscessus subsp. abscessus, a multiplex PCR assay, was developed using the whole-genome sequence of M. abscessus subsp. massiliense JCM15300 as a reference. Several primer sets were designed for single PCR to discriminate between the strains based on amplicons of different sizes. Two of these single-PCR target sites were chosen for development of the multiplex PCR assay. Multiplex PCR was successful in distinguishing clinical isolates of M. abscessus subsp. massiliense from samples previously identified as M. abscessus. This approach, which spans whole-genome sequencing and clinical diagnosis, will facilitate the acquisition of more-precise information about bacterial genomes, aid in the choice of more relevant therapies, and promote the advancement of novel discrimination and differential diagnostic assays.

Atkinsiella awabi sp. nov. isolated from stocked abalone,Haliotis sieboldii

A fungal disease in the abalone,Haliotis sieboldii, stocked in Yamaguchi Prefecture, Japan, showed external signs of infection of tubercle-like swelling on the mantle and melanized lesions on the peduncle. The fungus responsible was isolated by inoculating materials taken from the lesions onto PYGS agar with streptomycin sulphate and ampicillin, and incubation at 20°C. For morphological observation and spore formation study, the fungus was transferred respectively into PYGS broth and sterilized artificial seawater and incubated at 20°C. Resulting, hyphae were stout, irregular, branched, 16–140µm diam, sporadically consisting of dense cytoplasmic swollen hyphae. Sporangia were formed through the formation of septa and lateral or terminal discharge tubes which were wavy or coiled. Zoospores were pyriform, biflagellate and diplanetic. The encysted spore generally developed a hairlike filament with globular enlarged tip in PYGS broth. Direct germination without filament formation also occurred occasionally. This fungus was identified as belonging to the genusAtkinsiella, and was designatedAtkinsiella awabi sp. nov. The fungus was exclusively a marine fungus and grew best in shrimp extract medium at 20°C. Five chemicals were tested for their effects against fungal zoospores.

Cladosporiosis in a tomato clownfish (Amphiprion frenatus).

Abstract Infection with Cladosporium sp., a deuteromycete, caused a deep dermal ulcer that extended to bone in a cultured tomato clownfish (Amphiprion frenatus). The infection may have been secondary to immunosuppression resulting from transport or confinement-induced stress or may have resulted from chronic exposure to copper. Reports of fungal infections in tropical marine fish are rare.

Clinical observations of black disease in fairy shrimps, Streptocephalus sirindhornae and Branchinella thailandensis, from Thailand and pathogen verification

In this study, black disease infecting fairy shrimps, Streptocephalus sirindhornae Sanoamuang, Murugan, Weekers & Dumont, and Branchinella thailandensis Sanoamuang, Saengphan & Murugan, in Thailand, was investigated. The typical signs of the disease are the appearance of black spots on the cuticle, located mainly on the dorsal side and thoracopods. A number of rod-shaped bacteria aggregated in the black spots and were visualized by scanning electron microscopy. The histopathological results showed that a haemocytic response to the infection resulted in a dense melanized core of bacteria. In addition, generalized septicaemia by rod-shaped bacteria was also observed in the infected tissue. Of the 31 isolates, Aeromonas spp. were predominantly isolated and six strains were selected for the experimental infections. The most pathogenic strain was identified molecularly as A. hydrophila. When fairy shrimp were infected at bacterial concentrations of 10(4) and 10(6) cfu mL(-1) , the overall infection levels were 73.33 ± 6.67% and 93.33 ± 6.67%, respectively. The experimentally infected fairy shrimp showed abnormal swimming and died within 24-48 h after the appearance of the dark pigment.

Lymphocytes with T-cell-like properties express the Fas ligand in the Japanese flounder Paralichthys olivaceus.

In this study, we aimed to identify the leukocyte population that expresses Fas ligand (FasL) in the Japanese flounder (Paralichthys olivaceus). The transcriptional activity of FasL was examined for the first time in the fish leukocytes. Transcription of the FasL gene in flounder leukocytes was significantly increased by phytohemagglutinin (PHA) treatment. All the leukocyte populations we tested possessed binding activity for PHA, but this was especially high in the lymphocyte population. However, the lymphocytes consisted of two subsets showing heterogeneity with respect to PHA binding, with the high-binding subset being surface IgM-negative. We also found that only the lymphocyte population showed a significant increase in the expression of the FasL gene after stimulation with PHA. In addition, only the lymphocyte subset showing high binding to PHA showed conspicuous expression of the FasL gene. This subset also had a CD3γ/δ+, CD8α+ and IgM heavy-chain (-) phenotype. These results suggested that lymphocytes with T-cell-like properties are FasL-expressing cells in the Japanese flounder.

Exophiala xenobiotica infection in cultured striped jack, Pseudocaranx dentex (Bloch & Schneider), in Japan.

This report describes Exophiala infection in cultured striped jack, Pseudocaranx dentex, in Japan in 2005. One hundred out of 35,000 fish died per day and mortalities continued for 1 month. Diseased fish showed swelling of the abdomen and kidney distension. Numerous septate hyphae, pale brown in colour, were seen in kidney in squash preparations. Histology revealed abundant fungal hyphae and conidia in gill, heart and kidney. Fungal hyphae were accompanied by cell necrosis and influx of inflammatory, mainly mononuclear cells. The fungus isolated from the diseased fish had septate hyphae, pale brown in colour and 1.8-3.0 microm in diameter. Conidiogenous cells were conspicuous annellides, short or cylindrical or fusiform in shape. Conidia were one-celled, ellipsoidal with smooth walls, accumulated in balls at the apices of annellides that tended to slide down, 1.5-2.0 microm in width and 3.0-5.0 microm in length. The fungus was classified into the genus Exophiala based on its morphology and as Exophiala xenobiotica based on the sequences of the ITS 1-5.8S-ITS 2 regions of rDNA. This is the first record of this fungus in a marine fish.

Lagenidium myophilum infection in the coonstripe shrimp, Pandalus hypsinotus

A fungal infection occurred in juvenile coonstripe shrimps,Pandalus hypsinotus, cultured at Hokkaido Institute of Mariculture, Hokkaido, Japan. The fungus was identified asLagenidium myophilum, the same fungus that had previously been isolated from the abdominal muscle of adult northern shrimps,Pandalus borealis, and larvae of the coonstripe shrimp. Histopathologically, numerous nonseptate hyphae were observed in the lesions, and melanized hemocytes were present within the blackened areas. The optimum temperature for growth of the present strain was 25–30°C, and the optimum NaCl concentration for growth was 0.5–1.0%. Its biological characteristics were compared with those ofLagenidium myophilum isolated from diseased larval coonstripe shrimp and adult northern shrimp. The fungus was pathogenic toward shrimps of the genusPandalus, which live in deep sea areas. The fungus could infect shrimps at various stages, from larva to adult.

Mixed Infection of an Acid-fast Bacterium and an Imperfect Fungus in a Napoleon Fish (Cheilinus undulatus)

A Napoleon fish (Cheilinus undulatus) was infected with both an acid-fast bacterium and an imperfect fungus. This is the first report of an acid-fast bacterial infection in Cheilinus undulatus, and the first observation of an imperfect fungus in the swim bladder of a tropical marine fish.

Mycotic gastritis of juvenile ayu (Plecoglossus altivelis) caused by Saprolegnia diclina Type 1.

Histopathological and mycological studies were made on mycotic gastritis in juvenile ayu, Plecoglossus altivelis. The disease occurred 5 days after the transportation of fish from a hatchery pond to two rearing ponds of Tochigi Prefectural Fisheries Experiment Station, Tochigi, Japan. The pyloric region of the stomach was red. Numerous fungal hyphae penetrated into the stomach wall. Seven fungal isolates were obtained from the lesions. Three isolates were identified as Saprolegnia diclina Type 1. Based on histology, hyphae were most numerous in the pyloric stomach, but also penetrated abdominal adipose tissue, the pancreas, spleen, kidney, swim bladder, gonad, and liver. In some cases, the hyphae penetrated trunk muscles.