Three batches of Penaeus monodon (total of 66 shrimp) were tested for the presence of WSBV by 2-step PCR, in situ hybridization and dot blot analysis using a nucleic acid probe. The results of in situ hybridization and 2-step PCR were consistent and used as the standard for comparison. Except for a low number of false negatives, results of dot blot using either DNA or tissue homogenate from the gill, pleopod, eyestalk and hemolymph paralleled the results of 2-step PCR and in situ hybridization. A quick dot blot analysis was also developed, which took only 4-4.5 h to produce results. We conclude that dot blotting using tissue homogenate from the eyestalk provides a rapid, cost effective and convenient diagnostic method for WSBV detection in the field and for screening P. monodon broodstock.
Biochemical properties of coho salmon artificially infected with the virus causing erythrocytic inclusion body syndrome (EIBS) were examined. In experimental fish injected with a homogenate from the erythrocytes of moribund fish naturally infected with EIBS, inclusion bodies were observed in erythrocytes at 2-3 weeks postinjection and hematocrit values decreased significantly at 4-5 weeks post-injection. Blood phosphatidylcholine hydroperoxide values of infected fish were lower than those of control fish. Polyunsaturated fatty acids composition of hepatic phospholipids from infected fish was lower than that of controls, and an unknown peak appeared in the fatty acid chromatogram at 4 weeks post-injection. Although gill Na+-K+ ATPase activity was not affected by EIBS infection, renal Na+-K+ ATPase activity of infected fish decreased at 4-5 weeks post-injection. These results suggested that lipid peroxidation was not caused by EIBS infection directly, but that fatty acid metabolism of fish and renal Na+-K+ ATPase activity were affected by the viral infection.
Yellowtail Seriola quinqueradiata fed a non-fish meal diet and control fish fed a standard fish meal diet were reared in neighboring net-cages under the same natural environmental conditions. The growth and food consumption of the fish fed the non-fish meal diet (NFM fish) were similar to the fish fed the control diet (control fish). Mortality due to a natural infection with Pasteurella piscicida occurred among NFM fish, but not among control fish during 55 days of feeding experiment. Plasma total cholesterol, free cholesterol and phospholipid of NFM fish were significantly lower than those of control fish. This result indicates that the decrease in the levels of plasma lipid components may be a good indicator of reduced disease resistance in yellowtail.
Penaeid rod-shaped DNA viruses (PRDV) in tissue homogenates of the naturally infected shrimp were subjected to various chemical and physical treatments, and inactivation of the virus was examined by infection experiment. The infection was examined by the appearance of the virus-infected nuclei in stomach cuticular epidermis. Disinfectants: The virus was inactivated by 1 min contact with 0.62 mg/l of oxidant or 30% of ethyl alcohol, by 10 min contact with 1 mg/l of sodium hypochlorite, 2.5 mg/l of povidone iodine, 5 g/l of formalin or 25 mg/l of trimethylammoniummethylene chloride, and by 60 min contact with 5, 000 mg/l of sodium carbonate peroxyhydrate at 25°C. NaCl: The virus was inactivated with 25% NaCl solution within 24 h. Chloroform: The virus was inactivated with chloroform within 15 min. Heat: The virus was inactivated within 60 min at 50°C, 1 min at 60°C and 0.2 min at 70°C, respectively. Desciccation: The virus absorbed in a filter paper was inactivated within 3 h desciccation at about 26°C. U.V. irradiation: PRDV was inactivated with U. V. irradiation of 1 × 104 μW·sec/cm2.
Red sea bream iridovirus (RSIV) causes epizootic disease outbreaks involving mortality in cultured marine fishes in southwestern Japan. The reactivities of anti-RSIV polyclonal antibody and the monoclonal antibodies (MAbs) against RSIV to the RSIV isolates from various cultured marine fish species and diverse geographic regions were examined. All the isolates showed similar reaction patterns by an indirect immunofluorescence (IF) test with anti-RSIV serum. However, some MAbs reacted with restricted number of RSIV isolates. The antigenic relationship between RSIV and three iridoviruses associated with systemic infection in fish, epizootic haematopoietic necrosis virus (EHNV), the iridovirus isolated from sheatfish (SFIV) and the iridovirus isolated from grouper (GIV) was examined. Although cross reactivities were observed between RSIV and other fish iridoviruses by IF test or immunoprecipitation test using anti-RSIV serum, none of the MAbs against RSIV reacted with EHNV, SFIV or GIV infected cells.
The potential pathogenicity of strains of Vibrio splendidus biovar II, which were isolated from bacillary necrosis of triploid larvae of Pacific oyster Crassostrea gigas in a hatchery in western Japan, was investigated. The course of experimental infection with virulent strains of V. splendidus biovar II was very rapid in 5-day-old veliger larvae with disease signs apparent within 6 to 12 h after exposure of larvae at doses of 104 to 106 CFU/ml, and mortalities up to 100% were recorded in 24 h at 105 and 106 CFU/ml. Although all the tested stages of larvae were experimentally infected by virulent strains of V. splendidus biovar II, a later stage (17 days posthatching) was less susceptible than the earlier stages of development. Diploid and triploid larvae were almost equally susceptible to this pathogen.Extracellular products and intracellular components of the strains were lethal to larvae, but their lethality did not correlate with the virulence of live cultures. These results suggest that the ability to elaborate toxins is not the only virulence factor in the pathogenicity of V. splendidus biovar II. The ability of this pathogen to bring about significant mortalities in oyster larvae at densities of 104 CFU/ml and its long survival in seawater make this pathogen a potential threat to larval oyster productions in hatchery systems.
Morbidity and mortality of adult (2-and 3-year old) color carp were recognized during June and July in 1994 and 1995 at a culture pond in Niigata Prefecture, Japan. The clinical signs were characterized by severe hemorrhages on the body surface, in particular ventrally and in the region of the mouth. The causative agent was identified as a myxosporean parasite, Thelohanellus hovorkai, which parasitized the connective tissues in various organs of color carp. Histopathological examination revealed hemorrhages, edema, extensive inflammatory cell infiltrations and exfoliation of dermal epithelium in the cutaneous lesions, associated with spore dispersion from matured plasmodia. Oligochaetes (Branchiura sowerbyi) in the culture pond were found to be infected with the corresponding aurantiactinomyxon actinosporean, and the prevalence reached a maximum of 81% in June. We propose to designate this disease “hemorrhagic thelohanellosis of carp”.
Immersion in bath suspensions for extensive time periods has been reported to enhance the uptake of particulate antigen by rainbow trout, Oncorhynchus mykiss. In order to determine if prolonged immersion also enhances soluble antigen uptake, juvenile rainbow trout, weighing 15 g, were immersed in BSA solutions at 15°C using exposure periods ranging from 3 minutes to 48 hours. BSA uptake, measured as plasma concentration, was significantly dependent upon the exposure period (p < 0.001), demonstrating that prolonged immersion enhances the uptake of soluble antigen as well as particulate antigen. Furthermore, we found that BSA uptake was significantly dependent upon the BSA concentration in the bath solution during prolonged immersion. The plasma BSA concentration Cp (μg/ml) after immersion was related to time and concentration by the equation Cp (Cb, t) = 3.47·10-3·Cb·√t, where t is the exposure period in hours and Cb is the BSA concentration of the bath solution (μg/ml). The correlation coefficient of this formula is 0.99. Thus, the optimal combination of exposure period and antigen concentration in the immersion solution can be selected.