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.

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.

A new fluorochromasia method using a fluorescence microplate reader for assay of cytotoxic activity of carp leucocytes.

A new fluorochromasia method using a fluorescence microplate reader has been established to assay spontaneous cytotoxic activity of carp leucocytes. This method is characterized by using propidium iodide (PI) for staining dead target cells and a fluorescence microplate reader for measurement of the fluorescence of PI. K562 as target cells were prepared in 96-well flat-bottom microplates, and carp leucocytes were added as effector cells. After 2.5 h incubation, PI was added to each well. After an additional 1.5 h incubation, fluorescence of each well was measured. Correlation between this method and 51Cr-release assay was obtained. The results demonstrated that this new fluorochromasia method can be used to assay cytotoxic activity of carp leucocytes.

Neutrophilic granulocytes in carp, Cyprinus carpio, possess a spontaneous cytotoxic activity.

This study demonstrates for the first time that carp (Cyprinus carpio) neutrophilic granulocytes from the head kidney possess potent spontaneous cytotoxic activity against several human tumor cell lines. Carp head kidney cells isolated at a density of 1.09 g/mL contained more than 90% neutrophilic granulocytes. These cells were round and approximately 10 millimicrons in diameter with reniform or polymorphic nuclei and slightly eosinophilic cytoplasm when stained with Giemsa. Electron microscopy revealed that the cytoplasm contained numerous oval granules, some of which contained a dense rod-shaped core. The neutrophilic granulocytes readily formed conjugates with the human target cells and rapidly killed them. The neutrophilic granulocytes killed human derived target cells better than murine derived target cells. Inhibition of cytotoxicity by catalase suggested that the production of H2O2 is involved as a mediator in the cytotoxic reaction. The size and granularity of the carp effector cells indicate that they are different from the small agranular nonspecific cytotoxic cells (NCC) described in the channel catfish.