Red sea bream iridoviral disease (RSIVD) causes serious economic losses in the aquaculture industry. In this paper, we evaluated RSIV kinetics in rock bream under various rearing water temperatures and different RSIV inoculation concentrations. High viral copy numbers (approximately 103.7–106.7 RSIV genome copies/L/g) were observed during the period of active fish mortality after RSIV infection at all concentrations in the tanks (25 °C and 20 °C). In the group injected with 104 RSIV genome copies/fish, RSIV was not detected at 21–30 days post-infection (dpi) in the rearing seawater. In rock bream infected at 15 °C and subjected to increasing water temperature (1 °C/d until 25 °C) 3 days later, the virus replication rate and number of viral copies shed into the rearing seawater increased. With the decrease in temperature (1 °C/d) from 25 to 15 °C after the infection, the virus replicated rapidly and was released at high loads on the initial 3–5 dpi, whereas the number of viral copies in the fish and seawater decreased after 14 dpi. These results indicate that the number of viral copies shed into the rearing seawater varies depending on the RSIV infection level in rock bream.
The analytical and diagnostic performances of methods for detecting red sea bream iridovirus (RSIV), which infects marine fish, have not been evaluated. As disease management and transmission control depend on early and reliable pathogen detection, rapid virus detection techniques are crucial. Herein, we evaluated the diagnostic performance of a TaqMan-based real-time polymerase chain reaction (PCR) assay that detects RSIV rapidly and accurately. The assay amplified the RSIV, infectious spleen and kidney necrosis virus, and turbot reddish body iridovirus genotypes of Megalocytivirus and the detection limit was 10.96 copies/reaction. The assay’s performance remained uncompromised even in the presence of nine potential PCR inhibitors, including compounds commonly used in aquaculture. The variation of the cycle threshold values between assays performed by three technicians was evaluated using a plasmid DNA containing the major capsid protein gene sequence. The variation between replicates was low. The diagnostic sensitivity and specificity of the developed assay were evaluated using fish samples (n = 510) and were found to be 100% and 99.60%, respectively. Two technicians evaluated the reproducibility of the assay using fish samples (n = 90), finding a high correlation of 0.998 (p < 0.0001). Therefore, the newly developed real-time PCR assay detects RSIV both accurately and rapidly.
Red sea bream iridovirus (RSIV) is an important aquatic virus that causes high mortality in marine fish. RSIV infection mainly spreads through horizontal transmission via seawater, and its early detection could help prevent disease outbreaks. Although quantitative PCR (qPCR) is a sensitive and rapid method for detecting RSIV, it cannot differentiate between infectious and inactive viruses. Here, we aimed to develop a viability qPCR assay based on propidium monoazide (PMAxx), which is a photoactive dye that penetrates damaged viral particles and binds to viral DNA to prevent qPCR amplification, to distinguish between infectious and inactive viruses effectively. Our results demonstrated that PMAxx at 75 μM effectively inhibited the amplification of heat-inactivated RSIV in viability qPCR, allowing the discrimination of inactive and infectious RSIV. Furthermore, the PMAxx-based viability qPCR assay selectively detected the infectious RSIV in seawater more efficiently than the conventional qPCR and cell culture methods. The reported viability qPCR method will help prevent the overestimation of red sea bream iridoviral disease caused by RSIV. Furthermore, this non-invasive method will aid in establishing a disease prediction system and in epidemiological analysis using seawater.
Seahorses, which have been cultivated since the 2000s, are economically very important. Gas bubble disease (GBD) is a significant concern in the cultivation of seahorses; therefore, this study aimed to determine the cause of GBD-induced death in two species of Syngnathidae (Syngnathus schlegeli and Hippocampus haema). Rod-shaped bacteria were observed histopathologically and identified as Vibrio splendidus by conventional and real-time PCR analyses. The lethality of V. splendidus varies depending on the host's immune status, and the disease can be prevented through water quality management or improvement of the breeding environment. In this study, the GBD lesions (gas bubbles) were observed at 12℃, 8.0 mg/L of dissolved oxygen, 30 ppt of salinity, and pH 7.7. In addition, rod-shaped bacteria, infiltration of inflammatory cells, and extensive serous exudate were confirmed in the lesions where gas bubbles were found. PCR analysis was able to detect V. splendidus, possibly a secondary infection of the immunocompromised syngnathid fish. Understanding the risk of immunity control and the correlation between these lesions and causal agents will be of great help to the aquaculture industry and the ornamental fish market.
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