In southern part of Kyusyu, Japan, a disease with mortality has repeatedly occurred in the farmed yellowtail Seriola quinqueradiata and amberjack S. dumerili in the early summer since 1989. The affected fish, most of which weighed less than 100 g, had no specific external signs. After the dissection, reddening of the brain was noticed in all the diseased fish. Remarkable histopathological sign was meningitis across the brain and spinal cord in all the affected fish. However, no noticeable histological alteration was found in the visceral organ, gill and muscle. Bacteria-like particles were found in stamps of the brains. A Gram-negative, short-rod bacterium was isolated from the brain of all the diseased fish. Pathogenicity of the bacterium against yellowtail was confirmed by an experimental infection, but the taxonomy of this bacterium remains unknown. This is the first report on a bacterial infection in which the brain is the main target organ in a host fish.
Forty five strains of marine bacteria which showed inhibitory activities against bacterial swarming were isolated from larval black tiger shrimp (Penaeus monodon), brine shrimp (Artemia salina) nauplii and rearing seawater in Thailand. These marine bacteria were examined by a double-layer plate method for their growthinhibiting abilities against 50 strains of vibrios isolated from similar sources. As a result, 27 strains among 45 marine bacteria examined inhibited 32 to 94% of the vibrios tested. These Vibrio-inhibiting strains also exhibited inhibitory activities against 52 to 96% of the reference strains of 27 species of Vibrio including fish and shellfish pathogens. The inhibition was exhibited bactericidally, and antibiotics were produced extracellularly and inactivated by heat treatment at 70°C for 30 min. All the 27 strains of antibiotic producers were identified as Alteromonas-like organisms based on their taxonomical characteristics including G + C values of DNA (42.4-45.3 mol %).
The efficacy of praziquantel and mebendazole against Microcotyle sebastis infestation in juvenile rockfish by oral administration was evaluated at low (10 ± 1°C) and high (20 ± 1°C) water temperatures. Oral administration of praziquantel or mebendazole resulted in significant reduction or complete extermination of M. sebastis infestation in juvenile rockfish. The treatment efficacy of praziquantel 200 mg/kg B. W. at both temperatures (10°C and 20°C) and mebendazole 200 mg/kg B. W. at 20°C was 100%. Praziquantel was more effective against M. sebastis than mebendazole at 10°C. Our results strongly suggest that oral administration of praziquantel and mebendazole can be used practically in netpen-culture of rockfish as an effective control measure against M.sebastis infestations without imposing handling-related stress to fish.
An infection of Perkinsus (Protozoa : Apicomplexa) was found in the short-necked clam in Kumamoto and Hiroshima prefectures, the first record in Japan. Histology revealed a number of trophozoites with a vacuole, forming cyst-like structures, in connective tissues of the gill, heart, mantle, digestive gland and adductor muscle of the infected clam. The protozoan in the gill enlarged by a culture in Ray's fluid thioglycolate medium and was colored by Lugol's iodine solution. The nucleotide sequences of two internal transcribed spacers with 5.8 SrRNA region were PCR-amplified from DNA of prezoosporangia obtained by a culture of the infected clam tissue. These sequences were almost identical to those of Perkinsus atlanticus and P. olseni. These facts suggest that the Perkinsus protozoan which infected short-necked clam in Kumamoto and Hiroshima, Japan is P. atlanticus or closely related species. We designed a PCR method for diagnosis of this Perkinsus protozoan. The PCR diagnostic method indicated high infection rates (64∼94%) of the parasite in short-necked clam in both area.
Three different extraction methods were used for preparing major outer membrane protein (MOMP) from the cell membrane fraction ofEdwardsiella tarda EF-1 which was isolated from Japanese eelAnguilla japonica. Triton X-100 extracted many proteins, including a thick band at 97 kDa in SDS-PAGE. Sodium dodecyl sulfate (SDS) and sodium lauryl sarcosinate (Sarkosyl), however, extracted three MOMPs at 37, 40 and 43 kDa, suggesting that these three MOMPs are closely associated to the membrane. The mobilities of the 37, 40 and 43 kDa MOMPs in SDS-PAGE were not affected by 2-mercaptoethanol treatment. Native-PAGE of the SDS-extracted MOMPs showed an unseparated protein band only at high molecular weight range. These findings indicate that the three MOMPs compose large molecules without intramolecular disulfide bridges in the outer membrane ofE. tarda EF-1. These MOMPs were distinctly detected in the western blotting with rabbit antiserum against EF-1 whole cell, indicating the three MOMPs are involved in the determination of the cell antigenicity. SDS-PAGE profiles of the MOMPs prepared by SDS-method from 15E tardastrains, which were isolated from Japanese eel, yellowtail Seriola quinqueradiata, Japanese flounderParalichthys olivaceusand crimson sea breamEvynnis japonicain Japan, showed a major protein located at 37 kDa, although 2 strains from Taiwan showed a slightly smaller molecular weight size. This MOMP was visualized in western blotting by using the anti-Etarda EF-1 serum, with no cross reaction of MOMPs of bacterial species in other genera :Escherichia, Photobacterium, Pseudomonas, AeromonasandVibrio. The results indicate an epitope included in the MOMP located around 37 kDa is common amongE. tardastrains and specific to this species.
Hemorrhagic thelohanellosis in two varieties of carp (Cyprinus carpio) caused byThelohanellus hovorkai was investigated by experimental infection. One-year old coloured carp (koi-carp) and common carp were kept for five weeks in tanks with oligochaetes (Branchiura sowerbyi) collected from a pond where hemorrhagic thelohanellosis was enzootic. Another group of coloured carp was exposed only to the effluent from a tank containingB.sowerbyi. Four weeks post-exposure, both coloured carp and common carp maintained withB. sowerbyishowed clinical signs of infection and chronic mortalities. On the other hand, neither mortalities nor clinical signs were observed in the effluent group, though light infections withT. hovorkaiwere detected. Control groups of coloured carp and common carp maintained in clean tanks showed no signs of infection at all. Through a standardized trypsin digestion method, the intensity of infection withT. hovorkaiin carp maintained withB. sowerbyiwas determined to be more than 50, 000 spores/g-tissue. Histological observations revealed thatT. hovorkaiplasmodia were most frequently detected in connective tissues of the intestine and the ventral skin in the cohabitation group, whereas they were found most frequently in the gills of the fish of the effluent group. In conclusion, hemorrhagic thelohanellosis was experimentally induced in carp by maintaining hosts withB. sowerbyiregardless of host's varietal difference, and the ingestion ofB. sowerbyiinfected with the actinosporean may be an important factor inducing the disease conditions.
A new epizootic causing mass mortalities among cultured Japanese abalone, Sulculus diversicolor supratexta, occurred in Kanagawa Prefecture, Japan, during the period from June to October in 1997. Diseased abalone lost the ability to adhere and eventually died on the bottom of aquaria. White spots on the foot were a characteristic sign of the disease. In sections of tissues with lesions, there was necrotic degeneration in muscle fibers, and homogeneous bacterial cells were isolated from the infected tissue. Isolates of this bacterium were Gramnegative, facultatively anaerobic, rod shaped organisms that were motile by a single polar flagellum. They were identified as Vibrio carchariae on the basis of phenotypic characteristics, 16S rRNA gene sequences and DNA-DNA hybridization, although some phenotypic properties of the isolates differed from those of references. Infection experiments confirmed the pathogenicity of the bacterium to Japanese abalone. This is the first report of the isolation of V. carchariae from a diseased shellfish.
The present study was attempted to investigate the prevalence of penaeid rod-shaped DNA virus (PRDV) in wild-caught adult kuruma prawn Penaeus japonicus using a 2-step polymerase chain reaction (PCR) as a detection method. The prawns examined were captured in 5 different areas of coastal waters in Japan from July 1996 to April 1998. Individual samples of hemolymph, stomach and gonad of these wild female and male prawns were submitted to PCR test. As a result, PRDV was detected in female (10.1%, 96/955) and male (6.7%, 21/314) prawns captured in all coastal waters, although most prawns showed no white spot in the carapace. Higher detection rates were obtained from stomach, ovary and testis than from hemolymph in female and male prawns. The prevalence of PRDV in adult prawns showed a seasonal and sex variation, i.e. the prevalence in summer (July to August) was higher than in winter (December to January) in female, and the prevalence in female was higher than that in male prawns. These results suggest that wild-captured adult kuruma prawns, especially those collected in summer, become an infection source for vertical transmission of PRDV when they are used as spawners.