Tenacibaculum sp., the causative bacterium of spotting disease of short-spined sea urchin Storongylocentrotus intermedius, has been known to enter into the viable but non-culturable (VBNC) state in 75% Herbst's artificial seawater at 5°C. Previous studies indicated that elevation of water temperature or addition of iron induced the resuscitation of the bacterium from the VBNC state. The present study revealed that the resuscitation period from the VBNC state was further prolonged when Tenacibaculum sp. in the VBNC state was resuscitated under the presence of iron, catalase and the homogenate of the sea urchin. Environmental waters around a sea urchin hatchery in Hokkaido, Japan often contained iron at concentrations over 0.34 mg/L in summer. The infection experiment showed that the VBNC cells activated with iron produced the spotting disease to healthy sea urchins. These results suggest that the VBNC cells of Tenacibaculum sp. resuscitated during summer under iron-rich environment are associated with outbreaks of the disease.
In spring of 2003, a mass mortality occurred in a population of marbled sole Pleuronectes yokohamae reared at a hatchery of Miyagi Prefecture, Japan. Since the first case, outbreak of the disease has been repeated every year in the hatchery. The affected fish weighing 1-5 g showed hemorrhagic lesions on the body surface. The maximum cumulative mortality reached more than 80%. The prominent histopathological feature was hemorrhage and necrosis in the gill, muscle, liver, heart and kidney associated with extensive bacterial multiplication. A single species of bacterium was isolated from the kidneys of the diseased fish using trypticase soy agar containing 1% NaCl at 20°C. The isolates were identified as atypical Aeromonas salmonicida by biochemical, serological and molecular phylogenetic analyses. Experimental infections confirmed that an isolate was highly pathogenic not only to marbled sole, but also to Japanese flounder Paralichthys olivaceus and spotted halibut Verasper variegatus, with LD50 of less than 102 CFU/fish (1.4-6.8 g body weight) by intramuscular injection.
We examined the adhesion of Tenacibaculum sp., the causative bacterium of spotting disease of short-spined sea urchin Stronglycentroutus intermedius, to the host. The number of adhesive cells of Tenacibaculum sp. strain F-2 isolated from diseased sea urchin was about 30 times more than those of two stains of non-pathogenic marine Cytophaga sp. isolated from healthy sea urchins. The adhesion of Tenacibaculum sp. F-2 to the sea urchins was inhibited by about 90% when the sea urchins were pre-treated with 0.1% D-galactose or D-xylose for 1 h. With this treatment, all sea urchins remained asymptomatic and were still alive at 7th day after being immersed with 106 or 107 CFU/mL Tenacibaculum sp. F-2 for 1 h at 23°C, while mortality of control reached 100%. These results indicate that carbohydrate treatment of the sea urchin is useful to control the disease.
We designed a LAMP (loop-mediated isothermal amplificatio) primer set comprised with six primers from koi herpesvirus (KHV, CyHV-3) specific PCR amplicon (accession number: AF411803) and developed a rapid and sensitive detection method for KHV. The target sequence was amplified under isothermal condition at 65°C within 60 min, and the detection limit was at least six copies of the plasmid inserted KHV specific sequence. Cross-reactivity with other fish-pathogenic viruses and bacteria was not observed. The reaction was monitored in real-time by an increase in the turbidity of a large amount of by-product, pyrophosphate. As an alternative method, a visual detection at end-point of reaction could be achieved by an increase of turbidity in reaction mixture with naked eyes. The LAMP method was applicable to amplify the target sequence from crudely extracted samples of gills by a simple procedure.
The hemagglutinating activity (HA) of Edwardsiella tarda, which had been isolated from cultured fish and culture environments, was investigated in relation to NaCl concentration of the growth medium. E. tarda cells were cultured in a peptone-yeast extract broth supplemented with 3% NaCl (3%-NaCl culture) and without NaCl (0%-NaCl culture). Hemagglutination assays with guinea pig erythrocytes classified the strains into three HA patterns. Seventeen strains exhibited HA only with the 3%-NaCl culture (type A). A more frequent type (35 strains) displayed HA in both 0%- and 3%- NaCl cultures but the 3%-NaCl culture showed higher HA activity than the 0%-NaCl culture (type B). No HA was detected in both cultures of the other three strains (type C). The NaCl-induced HA was well correlated with the expression of a 19.3 kDa protein, a fimbrial major subunit (FimA). Infection experiments with a selected strain (type A) of E. tarda revealed that the 3%-NaCl culture was more virulent to Japanese flounder Paralichthys olivaceus than the 0%-NaCl culture, when fish were challenged by an oral route. This induction of the fimbrial protein under higher NaCl concentration may play an important role in the virulence of E. tarda in marine environments.
A fungal infection was found in eggs and larvae of black tiger shrimp Penaeus monodon at a hatchery in Chachensao Province, Thailand in August 2000. Fungi were isolated from eggs and larvae with fungal infection, and studied on the morphological and biological characteristics. When it was transferred from PYGS broth to artificial seawater, discharge tubes developed from the mycelia, and a vesicle for zoospore formation was produced at the top of each discharge tube. The characteristic feature of an asexual reproduction of the fungus was that zoospores swam away in seawater after the vesicle separated from the discharge tube. Based on these morphological characteristics, the fungus was identified as Lagenidium thermophilum. Some biological characteristics of the selected isolate NJM 0031 were compared with the other species in the genus Lagenidium isolated from some crustaceans. As a result, the isolate NJM 0031 showed similar characteristics to those of L. thermophilum ATCC 200318 isolated from mangrove crab Scylla serrata. The isolate was demonstrated to be pathogenic to larvae of black tiger shrimp by artificial infection. This is the first report of L. thermophilum infection in black tiger shrimp in Thailand.