Clonal structure in Ichthyobacterium seriolicida, the causative agent of bacterial haemolytic jaundice in yellowtail, Seriola quinqueradiata, inferred from molecular epidemiological analysis.

Bacterial haemolytic jaundice caused by Ichthyobacterium seriolicida has been responsible for mortality in farmed yellowtail, Seriola quinqueradiata, in western Japan since the 1980s. In this study, polymorphic analysis of I. seriolicida was performed using three molecular methods: amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Twenty-eight isolates were analysed using AFLP, while 31 isolates were examined by MLST and MLVA. No polymorphisms were identified by AFLP analysis using EcoRI and MseI, or by MLST of internal fragments of eight housekeeping genes. However, MLVA revealed variation in repeat numbers of three elements, allowing separation of the isolates into 16 sequence types. The unweighted pair group method using arithmetic averages cluster analysis of the MLVA data identified four major clusters, and all isolates belonged to clonal complexes. It is likely that I. seriolicida populations share a common ancestor, which may be a recently introduced strain.

Induction of specific cytotoxic T-cell activity against xenogeneic target cells in carp (Cyprinus carpio).

OBJECTIVE: To investigate the induction of cytotoxic T cells in carp (Cyprinus carpio) after inoculation of fish with 2 xenogeneic line cells and to examine specificity of the cytotoxic activity. ANIMALS: 22 carp. PROCEDURE: Fish were inoculated with mouse myeloma line cells P3.NS-1/1Ag4.1 (NS-1) or chicken Marek's disease tumor-derived lymphoma line cells (MDCC MSB-1). Cytotoxic activity of immune lymphocytes was evaluated by incubating effector cells with homologous and heterologous target cells. Populations of effector cells were identified by blocking T-lymphocytes from effector cells, using anti-carp T-cell monoclonal antibody and complement. RESULTS: Lymphocytes in blood, spleen, and head kidney of carp inoculated with NS-1 cells or MDCC MSB-1 cells had dose-dependent cytotoxic effects against homologous target cells but not against heterologous target cells. Lymphocytes from noninoculated carp did not have cytotoxic effects. Depletion of T-lymphocytes in spleen cells from NS-1-inoculated carp resulted in a decrease of cytotoxic activity against NS-1 cells. Cytotoxic activity of spleen lymphocytes from NS-1-inoculated or noninoculated carp was not evident when cytotoxic tests were performed after addition of anti-NS-1 carp serum. CONCLUSIONS AND CLINICAL RELEVANCE: Inoculation with xenogeneic target cells induces a specific cytotoxic T-cell response in carp. Thus, cell-mediated immunity plays a role in defense against infection of parasitic organisms such as protozoa and helminths.

Sensitivity of the olfactory sense declines with the aging in senescence-accelerated mouse (SAM-P1).

The decline in olfaction with age is well documented in histological, psychological, and electroencephalographical studies. However, there are few electrophysiological studies on changes in the sensitivity of the peripheral olfactory cells with age. We evaluated the behavior, the amplitude of electro-olfactogram (EOG), and the thickness of the olfactory epithelium in the Senescence-Accelerated Mouse (SAM-P1). This strain of mouse exhibits accelerated senescence and age-related pathologies, and it is commonly used as a model for research on aging. Its median survival time is 55 weeks. To ensure our results would be restricted to the olfactory system, we chose vanillin as a stimulus, because this stimulus has no definitive chorda tympani (VII) response, and we verified that it is tasteless. The data demonstrate that olfactory sensitivity to vanillin decreases dramatically with age in these mice, and that this is due to loss in the number of olfactory receptor cells.

Characterization of a monoclonal antibody specific to rainbow trout thrombocytes.

A monoclonal antibody (MAb) specific for rainbow trout thrombocytes was produced and its reactivity was demonstrated by flow cytometry and immuno-electron microscopy. Flow cytometry analysis showed that this MAb (TTL-7D11) reacted positively with about 30% of the peripheral blood leucocytes (PBL) and about 1%, 2%, and 11% of the pronephros, mesonephros, and spleen cells, respectively. Electron microscopy using immunogold labeling demonstrated that this MAb reacted strongly with thrombocytes, where gold beads could be seen attached only to the membrane and canalicular system of these cells. Positive and negative leucocytes for this MAb were obtained by magnetic cell separation. In the positive fraction, 96% of the cells were thrombocytes, while in the negative fraction no more than 3% were, which clearly showed a high purity of the positive fraction. Aggregation studies showed that about 75% of the positive fraction cells aggregated after being mixed with U-46619 thromboxane-mimetic, whereas in the negative fraction only 10% of the cells did so. Thus, utilizing the TTL-7D11 we have succeeded in isolating a pure thrombocyte population, and this would facilitate further studies, particularly on their characteristics and function(s).

Site-specific lead distribution in scales of lead-administered carp (Cyprinus carpio) by non-destructive SR-XRF analysis.

By utilizing non-destructive synchrotron radiation-excited X-ray fluorescence (SR-XRF), we observed the distribution of lead (Pb) in both ontogenic and regenerating scales of lead-administrated carp, Cyprinus carpio. The fish in the Pb-administered group were fed pellets containing 1 mg/g of Pb at a rate of 1.5% body weight per day for 30 days. In the ontogenic scales, Pb was highly accumulated near the basal edge of the scale and the accumulated amount decreased toward the focus of the scale. On the other hand, in the regenerating scales, high accumulation was observed near the basal edge and the accumulated amount remained high toward the focus. The present results of Pb accumulation correspond well with the region which is calcifying in the ontogenic and regenerating scales, and indicate that the distributions of Pb show when and how long Pb was administered.

Separation of carp (Cyprinus carpio L.) thrombocytes by using a monoclonal antibody, and their aggregation by collagen.

A monoclonal antibody against carp peripheral blood leucocytes was produced, and its reactivity analysed by flow cytometry and electron microscopy. The antibody reacted positively with 10-35% of the cells in a fraction of lymphocytes and thrombocytes that was separated by flow cytometry. Electron microscopy using immunogold labelling showed that this antibody reacted strongly with thrombocytes, but not with other leucocytes. By using a magnetic separator, leucocytes that were positive and negative for this antibody were separated. The positive cells were uniform, spindle-type cells that aggregated in the presence of collagen, while the negative cells did not aggregate. Light and electron microscopy showed that many positive cells changed to a spherical form after the addition of collagen and then 40-60% of these cells aggregated.