Seasonal surveys on aerobic bacterial flora in the digestive tract of 10 healthy Pacific oyster (Crassostrea gigas), which had been cultured in Ondo, Hiroshima Prefecture, were carried out in 4 seasons for 2 years.The number of bacteria in the digestive tract grown on ZoBell's 2216e agar was about 105 (104.3-105.6) CFU/g, and no statistically significant differences in the number were observed between seasons. The intestinal microflora was dominated by the genusVibriofollowed by generaCytophaga, Alteromonas, Moraxellaand Pseudomonaswhich occupied over 80% of the total flora in every season. AmongVibriospecies, five major species (V.splendidusI/II, V.harveyi, V.campbelliiand V.pelagius II) were predominantly isolated, especially V. harveyibeing most common (48%) in the summer. This species fluctuation and a result of anin vitrogrowth temperature test on the summer-isolated and the winter-isolated vibrios supported the presence of seasonal changes in dominant bacterial species in the oysters. An additional survey was tried on the hemolymph of 21 healthy oysters in winter, and one species of bacterium was isolated in a pure state on ZoBell agar at 103.6CFU/mL. This isolate was identified to the genusVibrioand confirmed to be virulent for adult oysters by an injection challenge.
The causative agent of amyotrophia in juvenile abalones, Haliotis spp., has not yet been identified. Infection experiments were undertaken using filtered homogenate of diseased abalones as an inoculum to examine the water temperature effect on the disease progress, infection titers of spontaneously and experimentally diseased juveniles and filterability of the pathogen. By water born inoculation, remarkable abnormal cell masses, the most characteristic histopathological change in amyotrophia, were observed in the nerve trunk and peripheral nerve in the foot muscle after 40 days and on, when inoculated juvenile Haliotis discus discus were reared at 18°C. When reared at 12°C, slight changes were observed in the animals even at the end of the test (after 60 days). At 24°C, although the cell masses were formed earlier than the other two water temperature groups, they tended to be restored after 40 days post-inoculation. Infection titers of the inocula prepared from spontaneously and experimentally (40 days after intramuscular inoculation) diseased juveniles were calculated to be 103and 105 ID50/mL/g flesh weight, respectively. The disease agent passed through 220 nm pore size filter, but not through 100 nm pore size filter.
To study IgM production in Japanese eel infected with Flavobacterium columnare, we measured serum IgM concentration (SIC), IgM constitution of total serum protein (ICT) and the number of antibody secretary cells (NASC) isolated from the blood, spleen and kidney. The fish were exposed to a bacterial suspension after removal of skin mucus in the tail region, and reared at 25°C. The values of SIC, ICT and NASC were significantly decreased 48 to 72 h after inoculation. When the eels were intraperitoneally injected with extracellular product (ECP) of the bacterium, a significant decrease in SIC, ICT and NASC isolated from the spleen was also observed 12 h after injection. In addition, ECP showed in vitro cytotoxicity against kidney lymphocytes of eel. It is considered that ECP is the major factor causing the decrease of IgM production.
An investigation on the distribution of Flavobacterium psychrophilum was conducted at the Umikawa River in Niigata Prefecture in 1999. Wild ayu (Plecoglossus altivelis), released ayu and other feral fishes (Oncorhynchus masou, Trybolodon hakonensis, Cottus kazika, Chaenogobius urotaenia, Rhinogobius brunneus, Tridentiger obscurus) were caught every month from May to December. In November newly-hatched ayu were caught in the river. In addition to fish, we collected water samples and small algae growing on the surface of stones on the river bed, which ayu lived on. By nested PCR and/or IFAT, the bacterium was detected in the gills and/or kidney tissue samples of both wild and released ayu as well as of four (Oncorhynchus masou, Trybolodon hakonensis, Chaenogobius urotaenia, Rhinogobius brunneus) of the six other fish species. The prevalence of the carrier fish was generally high in June and October. The eggs taken from wild mature ayu and the larvae collected in the river were revealed also to be carriers. Algae collected in May, November and December, and a water sample in December were found to be positive for F. psychrophilum by PCR. These findings suggest that non-ayu fishes as well as released ayu may serve as sources of infection for wild young ayu which migrate from sea to the river. It is also suggested that the coldwater disease may be transmitted from spawners to larvae.
We investigated the potency of two oil adjuvants, Montanidae ISA 763A (763A) and a squalene emulsion (Squalene), to enhance the response of ayu Plecoglossus altivelis to a formalin-killed bacterin (FKB) made from the coldwater disease etiologic agent, Flavobacterium psychrophilum. Ayu were challenged 4 wk after vaccination by an intramuscular injection with live pathogenic F. psychrophilum. Mortalities of fish injected with FKB of F. psychrophilum combined with either adjuvant showed that the adjuvanted vaccines had significantly higher (P<0.05) potencies than the FKB vaccine alone or sterile distilled water. Moreover, both adjuvanted vaccines produced significantly higher antibody titers than the FKB vaccine without adjuvant. From these investigations it is concluded that the use of Squalene and 763A adjuvants provides enhanced protection against coldwater disease in ayu compared to the use of FKB alone.
Four monoclonal antibodies (MAbs : Mm, MGm, MGcp1 and MGcp2) were produced against peritoneal exudate cells of rainbow trout, Oncorhynchus mykiss. The MAbs were characterized by immunofluorescence assay, flow cytometry, immunoelectron microscopy and Western blotting, and utilized to isolate macrophages and neutrophils by magnetic cell sorting. The immunofluorescence assay and flow cytometry showed that each of the MAbs was specific for both macrophage and neutrophil populations and these cells were observed in leucocytes from various origins, i.e. the head kidney, peripheral blood and peritoneal exudate. Mm and MGm reacted with membrane antigens, while MGcp1 and MGcp2 reacted with intracellular antigens. The MAb-positive cells were identified as both macrophages and neutrophils by electron microscopy using immunogold labelling. A Western blot analysis revealed that the MAbs detected different determinants (Mm; 205, 151 and 107 kDa : MGm; 205, 143, 107, 76, 55 and 52 kDa : MGcp1; no reaction : MGcp2; 64 kDa). Macrophages and neutrophils were efficiently isolated by a magnetic cell separation system with Mm and MGm. These antibodies will be useful for identifying and isolating rainbow trout macrophages and neutrophils, which facilitate studies of trout leucocytes.
0-year-old tiger puffer, Takifugu rubripes, were exposed to suspensions of Heterobothrium okamotoi oncomiracidia under different conditions. A fluorescent dye, CFSE (5 (6) -carboxy fluorescein diacetate, succinimidyl-ester), was used as a mean of labeling oncomiracidia to find them with a fluorescence Microscope immediately after settlement on the host. The labeling procedure did not affect the infectivity of the oncomiracidium. When fish were exposed to labeled oncomiracidia for 1, 3, 5 or 10 h, the settlement rate on the gills increased with increasing exposure time up to 3 h. When similarly exposed to labeled oncomiracidia of different ages (0-4 days after hatching), infectivity of the oncomiracidium decreased rapidly with increasing parasite age, and most oncomiracidia of 2 days or older failed to infect the gills. When exposed to 29, 58 and 116 labeled Oncomiracidia/ L/ fish and observed under fluorescence stereomicroscopy immediately after exposure, the settlement rates on the body surface and on the gills were not different among the 3 concentrations tested. The settlement rate on the body surface was always higher than that on the gills, irrespective of the difference in concentration. Thus, infection methods using tiger puffer and H. okamotoi oncomiracidia were standardized in terms of exposure time (3 h), parasite age (within 24 h after hatching), and parasite concentration (up to 116 oncomiracidia/ fish). CFSE-staining is suitable for the study of early stages of parasite settlement on the host fish.
Distribution of Pseudomonas plecoglossicida in tissues of ayu (Plecoglossus altivelis) challenged by immersion was studied by real-time quantitative polymerase chain reaction (Q-PCR). Fish were immersed in 107 CFU/mL of P. plecoglossicida suspension for 15 min. The fish were sampled at 1, 3, 6, 12, 24, 48, 72 h post-infection (p.i.) and the skin, gills, liver, spleen, kidney and blood were tested by Q-PCR. The skin and gills are likely to be portals of entry for the bacterium since P. plecoglossicida was found as early as 1 and 3 h p.i., respectively. The bacteria became concentrated in the liver, spleen and kidney at 6 h p.i., indicating that P. plecoglossicida can successfully invade ayu within 6 h p.i. Septicemia occurred at 48 h p.i., because high concentrations of the target DNA were abruptly found in the blood of infected fish. The sensitivity of QPCR used here was slightly higher than the culture method to identify subclinically infected fish by P. plecoglossicida. High throughput and turnover time of this assay is useful for monitoring large number of samples and rapid detection.