The study was conducted to develop a PCR and an improved PCR-RFLP analysis method for rapid species-identification of Aeromonas genospecies. The forward and reverse primers for PCR were designed from the complementary sequences of the 16S rDNA of all 15-recognized Aeromonas genospecies to amplify a 1206-bp PCR product. The PCR amplified the expected product from the DNA template of type or reference strains of all recognized Aeromonas genospecies as well as 106 Aeromonas strains, while no PCR product was obtained from any of the non-Aeromonas strains tested. The PCR-RFLP analysis using the restriction enzymes (Alul, Mbol, Pvull, Pstl and Narl) provided identification of almost all Aeromonas species. However, the Aeromonas sp. T8 group and A. caviae exhibited a similar RFLP pattern. Some selected biochemical tests were found to be helpful for differentiation of the two species.
Oncorhynchus masou virus (OMV) was isolated from naturally diseased rainbow trout Oncorhynchus mykiss cultured in Nagano Prefecture. In the present study, histopathological and electron microscopic features were performed on spontaneously and experimentally infected fish with OMV. The definite change was necrosis of OMV-infected cells, which were observed in the spleen, hematopoietic tissues in the kidney, liver, intestine, heart, gill filaments, epidermis and lateral musculature. In particular, the intestine showed severe necrosis and hemorrhage in the epithelium and underlying tissues, which is the first description of OMV disease.
Blood feeding of the monogenean Heterobothrium okamotoi from tiger puffer Takifugu rubripes was examined. Physiological saline containing fluorescent microspheres (1μm in diameter) was injected into the hepatic vein of one-year-old tiger puffer experimentally or naturally infected with H. okamotoi, and blood was taken from the host at 3 and 12 h post-injection (PI). Parasites were collected at 12 h PI and individually digested with KOH. Microspheres in the host blood and parasite body were counted by fluorescence microscopy. The amount of blood taken by individual parasites was calculated by the number of microspheres in the parasite body divided by the mean value of the microsphere concentration in the host blood at 3 and 12 h PI. The results show that the amount of blood increased as the parasite developed and that anadult fed 0.69±0.93μL of host blood for 12 h (=42μL/month). This is the first report of a quantitative study on the blood feeding of polyopisthocotylean monogeneans.
The myxosporean emaciation disease has been recently found in Japanese flounder Paralichthys olivaceus cultured in land-based tanks in Miyazaki Prefecture, Japan. Affected fish showed external signs of distended abdomen, rectal prolapse, sunken eyes and a typical bony ridge on the skull. Parasitological and histopathological examinations revealed that the developmental stages of Enteromyxum Ieei parasitized the intestinal epithelium of diseased fish. No other enteric myxozoans were observed in the affected fish. A molecular analysis of SSU rDNA confirmed that the causative myxozoan was identified as E. leei. Experimental transmission of E. leei was achieved to naive Japanese flounder and tiger puffer Takifugu rubripes by feeding with gut tissues taken from infected Japanese flounder. The present study indicates that the myxosporean emaciation disease of Japanese flounder is caused by E. leei.
Effectiveness of low rearing density was examined against red sea bream iridoviral disease (RSIVD) in juvenile red sea bream Pagrus major. In an experimental challenge test, in which the number of fish and the volume of rearing space were set in three levels, low-density groups (2.1 or 2.2 kg/m3) showed significantly lower mortalities than medium- (4.2 kg/m3) and high-density groups (8.4 kg/m3). In a field trial, fish were reared in neighboring net pens (6.4 m3) at three densities (0.8, 2.8, 9.6 kg/m3) for 73 days. The mortality due to natural infection with RSIV showed a clearly inverse correlation with the rearing density. Additionally, hemoglobin content and plasma component levels were measured in the before-mentioned field trial and an indoor experiment, in which the number of fish and the aquarium size were set in three levels (2.8, 5.3 or 5.8, 11.3 or 11.6 kg/m3). The levels of plasma total cholesterol and phospholipid in high-density groups were significantly lowered than those of low-density groups. Low rearing density was effective against RSIVD in red sea bream, and plasma lipid components may play a role in the disease resistance.
This study evaluates the potency of aqueous and ethanolic decoction (individual extract) and concoction (mixed extract) of three common medicinal herbs i.e. Curcuma longa, Ocimum sanctum and Azardirachta indica to inhibite the in vitro growth of A. hydrophila. Among the decoctions A. indica exhibited the most potent antibacterial property against A. hydrophila. Among the concoctions both the aqueous and ethanolic tri-herbal extracts mixed in the ratio of 1 : 1 : 1 have the highest antibacterial activity even at the lowest concentration than the other concoctions and decoctions.
The long-term maintenance of primary cell cultures from ovarian tissue of kuruma prawn Penaeus japonicus was examined in modified Leibovitz-15 medium supplemented with four growth factors (epidermal growth factor, basic-fibroblast growth factor, insulin and ciliary neurotrophic factor) and an antioxidant (2-mercaptoethanol; 2-ME). This medium allowed the primary-cultured cells to survive for a long period up to six months, while only for 45 days in the absence of the five supplements. Efficacy assessment for each supplement revealed that only 2-ME facilitated long-term maintenance of cell sheets.
Eight Fusarium strains were isolated from five prawns showing black gills at a farm in Kagoshima Prefecture, Japan in December 2001. On the other hand, no fungi were isolated from five prawns without black gills. Two out of the eight strains were morphologically identified as F. oxysporum and the other six strains as F. solani. Morphological characteristics of F. oxysporum were described and illustrated. The fungus showed pathogenicity when injected to juvenile kuruma prawns. This is the first case of F. oxysporum infection of kuruma prawn in Japan.
A polymerase chain reaction (PCR) method was developed to detect Myxobolus acanthogobii, a myxozoan parasite causing skeletal deformities in marine fishes. The PCR system targeting SSU rDNA of M. acanthogobii was confirmed to be species-specific; the detection limits of a single-and a nested-PCR were 1 and 0.01 pg DNA, respectively. Nested-PCR analyses were applied to epizootiological surveys of M. acanthogobii in cultured Japanese mackerel Scomber japonicus and in feral fishes (37 species). Three wild fishes, forksnout searobin Lepidotrigla alata, crimson seabream Evynnis japonica and scribbled toby Canthigaster rivulata, were found to be the hosts for M. acanthogobii.
Koi herpesvirus disease (KHVD) is an emergent cyprinid herpesvirus disease causing mass mortalities in koi and common carp Cyprinus carpio worldwide since 1998. In Japan, KHVD broke out in Autumn 2003 and has caused mass mortalities in farmed fish as well as wild fish all over Japan. Establishment of control measures against KHVD is the urgent theme, and in order to address this issue many scientists belonging to universities, national and prefectural research institutes, and pharmaceutical companies have studied on KHVD and produced excellent results. Not only scientists but also fish farmers are eager to know the results. This context motivated the author to organize a symposium in Mie. The symposium focused on the up-to-date results of studies on KHVD: KHV genome analysis, methods to inactivate KHV in the environmental water, ways to coexist with KHV, development of a delivery system for oral vaccination using liposome, and successful oral vaccination with liposomes entrapping KHV antigens.