Several Australian betanodaviruses (NNVs) and other, exotic NNVs were used in a study of the performance of a nested PCR test in common use in Australia and New Zealand. This test for the detection of NNVs, designated the ANZSDP VNN Nested PCR, was compared with a currently available commercial PCR kit for NNV detection. With respect to the endemic viruses, the ANZSDP was pan-specific for the NNVs tested, while the commercial PCR kit failed to detect the South Australian strain of barramundi Lates calcarifer NNV. With respect to the relative sensitivities of the two tests, in general, the ANZSDP appeared more sensitive than the commercial test. The exception was the Australian bass Macquaria novemaculeata NNV isolate, which was detected at lower levels by the commercial test. Among the exotic NNVs, both tests detected red-spotted grouper nervous necrosis virus and barfin flounder nervous necrosis virus, but neither test detected striped jack nervous necrosis virus (SJNNV). Further investigation revealed mismatches between SJNNV sequences and the nested primers used in the ANZSDP.
We examined the therapeutic effect of Streptococcus iniae phages isolated from fish culture environments against experimental streptococcicosis of Japanese flounder Paralichthys olivaceus. Phage sensitivity tests with a double agar method revealed that 31 of 35 S. iniae strains from the flounder have a similar sensitivity to six phage isolates. In phage therapy experiments, fish were injected intraperitoneally (IP) with S. iniae PSi402 and 1 h later IP-injected with a mixture of two or four phage isolates, and observed at 25°C for 2 wk. Mortalities of fish receiving phages were significantly lower than those of control fish without phage-treatment in all four trials. The effect of phage treatment was also demonstrated even at 24 h post-infection, when cell numbers of S. iniae were 107.4 and 104.5 CFU/g in the kidneys and brains of fish, respectively. However, as phage-resistant S. iniae were frequently isolated from dead fish in the phage-treated group, further investigations are required to establish phage therapy of the disease.
To isolate the causative virus for viral endothelial cell necrosis of eel (VECNE), we established the cell line JEEC, originated from vascular endothelial cells of Japanese eel Anguilla japonica. CPE with hypertrophied nuclei was found in JEEC inoculated with the filtrate of homogenized gills of diseased fish 7 days after inoculation. In the hypertrophied nuclei, icosahedral virus particles of about 75 nm in diameter were observed. The nucleic acid in the isolated virus was DNA. The virus was tolerant to chloroform and pH 3.0. It showed tolerance up to 42°C. Thus, this virus was classified as an adenovirus. The experimental infection by injecting the virus at 105.75 TCID50 into the abdominal cavity of healthy eel produced congestion in the central venous sinus of gill lamella, which is the typical sign of this disease, with the cumulative mortality being 60%. The virus was recovered from the gills, liver and kidney of the experimentally infected fish. Therefore, the virus of VECNE was identified as the causative agent.
We investigated the presence of Flavobacterium psychrophilum in the ovarian fluids of female adults (eight rivers) and the fry kidney tissues (nine hatcheries) of chum salmon Oncorhynchus keta in Hokkaido, Japan in 2005. F. psychrophilum was detected by culture in 3.3-90.0% of ovarian fluid at all rivers and 5% of kidney tissue at one hatchery, whereas 33.3-100% and 5.0-42.5%, respectively, at all the sampling sites were positive in nested PCR targeting 16S rDNA of F. psychrophilum. Isolated bacteria were identified by an agglutination assay, the nested PCR assay, biochemical tests and PCR targeting gyrB gene. Intraperitoneal injection with 5.3 × 105 to 3.9 × 106 CFU of the isolates/g to chum salmon, rainbow trout O. mykiss and masu salmon O. masou juveniles (5.3-6.4 g) resulted in cumulative mortalities of 10-70%, 4-26%, and 20-95%, respectively. These results suggest that the bacterium is widely distributed at high frequency in adult female chum salmon in Hokkaido and virulent to salmonid fishes.
The present study suggests that the myxosporean emaciation disease by enteric infection of Enteromyxum leei disrupts intestinal water uptake of tiger puffer Takifugu rubripes. This idea is based on a significant negative correlation between plasma chloride concentration and condition factor in diseased fish, and significantly higher osmolarity of plasma and major ion concentrations of the intestinal fluid in infected fish than in healthy control fish. Additionally, surgical ligation of the junction between the stomach and the intestine of healthy fish resulted in a 24% drop of body weight within 50 h, and significantly lower water content of the white muscle (operated fish 74 ± 0.5%; sham-operated fish 82 ± 1%). However, in vitro water uptake by isolated intestine sacs was not significantly different between the control and infected fish. Meanwhile, hepatic function appeared to be impaired as evidenced by the significantly lower hepato-somatic index (control fish 8.7 ± 0.8%; infected fish 2.7 ± 0.8%). Plasma activities of LDH, AST and ALT were all significantly lower in the infected fish. We propose that rapid loss of body weight of infected tiger puffer is mainly due to osmoregulatory failure but probably malnutrition is also involved in the pathogenesis.
Fingerling humpback grouper Cromileptes altivelis, experimentally infected with a betanodavirus (RGNNV genotype), were kept at 27°C, 31°C or 35°C for 14 days. The numbers of dead fish in ten fish at each water temperature were five at 27°C, two at 31°C and one at 35°C. The coat protein gene of the virus was detected by RT-PCR from four survivors at 27°C, two survivors at 31°C, but not from survivors at 35°C. Histopathologically, vacuolation in the retinal tissues wasobserved in three survivors at 27°C, but not in survivors at 31°C or 35°C. These results suggest that high water temperature inhibits the viral proliferation in fish.
In many yellowtail Seriola quinqueradiata farms in the vicinity of Uwajima city, Ehime Prefecture, Japan, a new disease has caused heavy mortalities of yearlings, mainly in summer and autumn. Symptoms included anorexia, lethargy with occasional bursts of swimming and redness in the central nervous system. In a histological survey, the most noticeable sign was severe encephalomyelitis. It is suspected that densely packed parasite-like cells (about 4 μm in diameter) in the spinal cords are the causative agent of this disease.
Among the four types of betanodaviruses, redspotted grouper nervous necrosis virus (RGNNV) has the highest optimum temperature (25-30°C) for its multiplication. We tested 16 RGNNV isolates for their temperature sensitivity in cultured cells and demonstrated that their upper temperature limits ranged from less than 30°C to 35°C. At the temperatures over the upper limits, viral RNA replication was inhibited similarly. These results indicate that temperatures mainly affect RNA replication or earlier virus multiplication processes. The incompetence of betanodaviruses at 37°C suggests their avirulence in human.