In the course of experimental infection of Penaeus monodon with white spot syndrome virus (WSSV) for virus isolation and purification, one unexpected virus was found to be dominant in purified virus samples prepared from shrimp hemolymph. Under electron microscopical observation, the virus was enveloped, rod-shaped and measured 140-200 nm×35-50 nm. Pure virions with a density of 1.154-1.162 g/mL in sucrose gradient were obtained and at least three viral structural proteins of 20, 63 and 110 kDa were observed when analyzed by SDS-PAGE. Injection of P. monodon with this purified virus demonstrated rapid and mass mortality. By ordinary histological studies, the cells with densely basophilic inclusions were found in the lymphoid organ, gill, stomach, hepatopancreas and heart. By electron microscopy, ultrathin sections of lymphoid organs from infected shrimp showed that numerous enveloped virions and nucleocapsids scattered or enclosed in the vesicles within the cytoplasm. The morphology, histopathology and structural proteins of this virus closely resemble those of the yellow head virus (YHV) from Thailand. Moreover, the results of RT-PCR diagnosis using primers specific to the 135 bp of YHV also suggest that this virus is closely related to YHV. PCR diagnosis of YHV and WSSV in P. monodon sampled from culture farms in Taiwan between 1996 and 1999 demonstrated that YHV has been present in P. monodon for at least 3 years and that all of the samples, which were diagnosed as YHV positive, were also co-infected with WSSV.
The amyotrophia of Japanese black abalone Nordotis discus discus, an infectious disease caused by an unknown filterable agent, is the most serious problem in abalone hatcheries in western Japan. In the present study, it was experimentally demonstrated that the disease agent was transmitted through water to susceptible 0-year-old abalones from convalesent 2-year-old carriers which had previously been produced by immersion challenge. The agent was also transmitted via water to juvenile abalones from female adults which were caught in the sea as brood stocks and suspected to be inapparently infected. These results indicate the importance of prevention of horizontal transmission of the amyotrophia agent in abalone hatcheries.
To test whether NADPH oxidase-like activity in hemocytes of the Pacific oyster, Crassostrea gigas, occurs, we investigated molecular oxygen (O2) consumption and superoxide anion (O2-) generation during the respiratory burst. When oyster hemocytes were stimulated with phorbol myristate acetate (PMA), a rapid increase in O2 consumption was recorded accompanying a cyanide-independent respiratory burst using a Clark-type oxygen electrode. The O2 consumption was almost completely inhibited by the addition of 2μM diphenyleneiodonium (DPI), a specific inhibitor of mammalian NADPH oxidase. Oyster hemocytes, on stimulation with PMA, exhibited a relatively strong O2--dependent chemiluminescent (CL) response at the peak photocount of 4.67×105CPM/2 × 106hemocytes. The CL response induced by PMA-stimulated hemocytes was markedly reduced by 88% in the presence of 2μM DPI. Furthermore, O2-generation was measured by the reduction of acetylated ferricytochrome c. When the primary O2-generation by PMA-stimulated hemocytes was terminated by detergent mediated cell lysis, and reconstituted by the addition of exogenous NADPH, O2-generation in the hemocyte lysate was restored. Findings obtained in our experiments suggest that an NADPH oxidase-like activity, which is associated with a specific O2--forming system similar to that in mammalian phagocytes, exists in oyster hemocytes.
In Taiwan, high mortality of cultured Pacific white shrimp Penaeus vannamei juveniles occurred within 3 days of disease onset in March, 1999. Moribund juveniles were anorexic and lethargic, exhibited ataxic swimming behavior, and had opaque musculature, a soft shell and a pale reddish coloration that made the tail fan and pleopods distinctly red. Survivors were found with multifocal, melanized, cuticle lesions on the cephalothorax and abdominal exoskeleton. Necrosis of the cuticular epithelium was found upon histopathological examination of moribund shrimp. Prominent necrosis in the cuticular epithelium and subcutis were found throughout the appendages, cephalothorax and foregut. The necrotic cells had pyknotic nuclei and cytoplasmic spherical inclusions, which ranged from eosinophilic to darkly basophilic. The cytoplasmic inclusions, and pyknotic and karyorrhectic nuclei gave lesions a peppered, or buckshot-riddled, appearance. In situ hybridization analyses using Taura syndrome virus (TSV) -specific cDNA probes showed that 87% of the juveniles examined were TSV positive and that cuticular epithelium was the target tissue. Negative staining of the purified viral preparations revealed the presence of icosahedral particles, 28 to 31 nm in diameter. These results indicate that TSV, or a very similar strains of the same virus, is responsible for the acute mortality of white shrimp in Taiwan. TSV may have been introduced to Southeast Asia with chronically infected brooders or asymptomatic carriers imported from the Western Hemisphere.
Fertilized coho salmonOncorhynchus kisutcheggs were immersed in diluted broth culture of Flavobacterium psychrophilum(1.0×108, 1.0×106, 1.0×104CFU/mL) for 30 min before water-hardening, after water-hardening and at eyed-egg stages. Just after exposure to the bacteria, the eggs were disinfected with povidone-iodine, and then discretely incubated in flowing water. After 7-28 days of incubation, F. psychrophilumwas isolated only from the egg group which had been exposed to 1.0 × 108CFU/mL before water-hardening. No bacteria were detected from the other egg groups exposed to 1.0 × 106or 1.0 × 104CFU/mL as well as the groups exposed to 1.0 × 108 CFU/mL after water-hardening or the group at the eyed-egg stage. The viable counts ofF. psychrophilumin the contents of the infected eggs ranged from 103to 107 CFU/g. Observations on the frozen sections stained by indirect immunofluorescent antibody technique revealed that manyF. psychrophilumcells were located within the infected eggs at the eyed stage. It was concluded that F.psychrophilumentered the eggs during the water-hardening stage.
This study demonstrates that the fish pathogenicAphanomyces piscicida (=A.invadans) possesses hemagglutinating and hemolytic activities. These activities were observed against goldfish erythrocytes using supernatant of the homogenized hyphae ofA. piscicida. The hemagglutinating activity was not inactivated by heat treatment at 60°C for 30 min or by the addition of EDTA. D-galactose or lactose inhibited the hemagglutinating activity. D-galactose-binding substances which were isolated by affinity chromatography showed hemagglutinating activity. On the other hand, heat treatment at 60°C for 30 min reduced the hemolytic activity. It was demonstrated that a sample that did not absorb to D-galactose in affinity chromatography was responsible for the hemolysis. The factors that have hemagglutinating and hemolytic capacities may affect the blood cells of infected fishin vivo, and may play a role in the occurrence of anemia in infected fish.
Based on the nucleotide sequences of 16S rDNA, Japanese isolates of atypicalAeromonas salmonicidafrom different fish species can be divided into four groups; isolates from each species of fish (including fish reared in similar culture conditions) always belonged to the same group. Isolates from goldfishCarassius auratushad sequences identical to those from goldfish in the USA and formed the first group. Isolates from Japanese eelAnguilla japonica and marine fishes (greenlingHexagrammos otakii, Japanese flounderParalichthys olivaceus, shotted halibutEopsetta grigorjewi and Schlegel's black rockfish Sebastes schlegeli) formed the second group, and had identical sequences with those ofA. salmonicidasubsp.achromogenes JCM7875T, A. salmonicida subsp. masoucida JCM7873TandA. salmonicida subsp. smithia ATCC49393T. The third and fourth groups composed of isolates from coloured carpCyprinus carpioand common carpC. carpio, respectively. This indicates that the expansion of ulcerative disease in recent years is caused by strains which have phyletic lines different from goldfish isolates.
To elucidate the process of the uptake of suspended particles into the skin, fins and gills of rainbow troutOncorhynchus mykiss, 100 fingerlings were immersed in a suspension of 1μm diameter fluorescent microspheres and rinsed with water. The fate of microspheres adhering to body surface was traced for elapsed times ranging from 1 min to 24 h. Although most microspheres were removed in a 2 h-rinse with water, a few were observed by light microscopy to be located in both epidermal and dermal tissues which had microscopic injuries. As observed by electron microscopy, microspheres were taken up by migrating epithelial cells, or embedded in the dermis covered with these cells. Some of those microspheres embedded in dermis were endocytosed by macrophages. Such features were confirmed in artificial wound experiments. We conclude that microscopic injuries to trout could be an important route for the uptake of antigen administered by immersion, 2 to 24 h after fish suffer from injuries.
A simple method for quantification of sodium nifurstyrenate (NFS) in a small amount of yellowtail serum was developed by use of high performance liquid chromatography. NFS was extracted with acetonitrile from serum added with NFS in vitro, injected directly into a C18 column and detected at 420 nm wavelength with 50 mM potassium phtalate (pH4) -acetonitrile (40 : 60) as a mobile phase. This method gave the average recoveries of 102% and linear relation between peak area and NFS amount. It was possible to determine the concentration of NFS from 2.5 ng to 1000 ng in 100μL serum.