Betanodaviruses, the causative agents of viral nervous necrosis of fish in aquaculture, can be genetically classified into four types, designated SJNNV, RGNNV, TPNNV and BFNNV. To investigate betanodaviruses in apparently healthy wild fish, we tested 20 fish species from some surrounding ocean areas of Japan for the detection of viral RNA using PCR-based methods. Of 729 fish tested, six (six species) and 87 (11 species) samples were positive for RT-PCR and nested PCR, respectively. These positive species have never been reported as susceptible hosts. Sequencing of obtained PCR amplicons revealed that RGNNV and SJNNV existed in nine and six fish species, respectively. BFNNV was found from one species, round herring Etrumeus teres. Interestingly, the RNA2 sequences thus obtained had more variations than those from wild fish caught around aquaculture facilities in our previous report. These results suggest that genetically varied betanodaviruses are widely spread around Japan.
Detection limit of PCR was investigated for Renibacterium salmoninarum, the causative agent of bacterial kidney disease, with a logarithmic growth phase. When genomic DNAs from serially diluted bacterial cells were subjected to PCR, the minimum concentration by PCR-detection with 30, 35 and 40 cycles of amplification were all 105 cells/mL, and the sensitivity was not improved by treatments of the bacteria with lysozyme, achromopeptidase and/or SDS. The extraction rate of genomic DNAs from the bacterial cells was approximately 3%. It was confirmed that PCR-detection limit for R. salmoninarum cells mixed with fish kidney tissues was significantly lower than that for the bacteria suspended in the medium. Moreover, there was no significant difference in the detection limit between immunofluorescence antibody technique (IFAT) and PCR. These results suggest that PCR detection should be followed by other methods such as IFAT.
A dematiaceous fungus was isolated from Japanese flounder Paralichthys olivaceus with ulceration and erosion of the skin surface. The fungus was identified as an Exophiala species, with different morphological, biological and molecular characteristics from three previously described pathogenic Exophiala species. Fungal hyphae extended laterally in the dermis, and were absent from the epidermis and musculature of the skin lesions and kidneys of the diseased fish. An inflammatory response with granuloma occurred in the dermis involving accumulations of epithelioid cells around the hyphae. The granulomas were surrounded by lymphocyte-like cells. Epidermal degeneration was observed above the inflamed dermis, suggesting that the inflammatory response caused epidermal damage. Experimental infection reproduced hyphal extension and infiltration of inflammatory cells in the dermis of the flounder, confirming the pathogenicity of the fungus.
Myxosporean emaciation disease of cultured red sea bream Pagrus major and spotted knifejaw Oplegnathus punctatus has recently occurred in Japan. Morphological features and molecular analysis of SSU rDNA indicated that myxozoans from the intestine of affected fishes were identified as Enteromyxum leei, one of the causative organisms of the emaciation disease of tiger puffer Takifugu rubripes. A one-year periodic examination of E. leei infection in cultured red sea bream revealed that cumulative mortalities reached about 10% during the first summer, but surviving fish were not infected in the following year. Experimental transmission of E. leei from infected tiger puffer to naive red sea bream was achieved by both cohabitation with infected fish and exposure to effluent from a tank containing infected fish. This study suggests that fish-to-fish transmission occurs among different fish species in culture fields.
A mass mortality of cultured ayu Plecoglossus altivelis occurred from May to June in 2006 in Hiroshima Prefecture. Most of the diseased fish showed exophthalmos, reddening of the head and curvature of the body. A Gram-negative bacterium was isolated from the brain and kidney of affected fish by using a blood agar medium, but not by non-blood agars. The bacterium was well-cultivable on non-blood agars supplemented with hemin at 25 μg/mL or higher concentrations. The bacterium was classified into the genus Vibrio but differentiated from any known vibrios, based on the results of conventional characterization tests and sequence analyses of 16S rDNA and the non-coding region of the intergenic transcribed spacers between 16S and 23S rDNA. Intraperitoneal injection confirmed the pathogenicity of the bacterium to ayu (mean body weight: 14.8 g) with the LD50 at 1.4 × 103 CFU/fish. Bacteriological and histopathological examinations on the experimentally infected fish revealed that the causative bacterium has high affinity to the brain of ayu.
Infection with the lernaeopodid copepod Clavella parva was found in gold-eye rockfish Sebastes thompsoni broodstock kept at an aquaculture institute in Aomori Prefecture, northern Japan. A description of female and male adults of C. parva is given. This is the first record of C. parva from Japan and a new host record. The parasite was likely introduced to the institute by the fish that had been reared in cages kept in natural waters. No infection was observed again after manual removal of the copepods. The species occurred most abundantly on the dorsal and caudal fins, followed by the anal fin. Literary information on the geographical distribution and hosts of C. parva is reviewed.