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The oomycete Aphanomyces invadans causes epizootic ulcerative syndrome (EUS), a World Organization for Animal Health (WOAH)-listed disease that has seriously impacted a wide range of fish worldwide. Currently, only three conventional polymerase chain reaction (PCR) assays are recommended for the detection of A. invadans. The robust quantitative PCR (qPCR) assay has recently become more important due to its highly accurate nature and the applicability of qPCR-based environmental DNA (eDNA) detection in the monitoring of pathogens in aquatic environments. Therefore, in this study, we developed a novel TaqMan probe-based qPCR method to sensitively and quantitatively detect A. invadans. The assay limit of detection was determined using 10-fold serial dilutions of linearized A. invadans plasmid. Assay sensitivity was assessed in the presence of interfering substances and compared to three WOAH-listed primers using the mycelia and zoospores of A. invadans with and without fish muscle tissue. The assay specificity was also theoretically and experimentally assessed against other oomycetes, fish muscle tissue, and water samples. The assay's repeatability and reproducibility were determined. In this study, the limit of detection of the developed assay was 7.24 copies of A. invadans genomic DNA per reaction (95% confidence interval (CI): 2.75 to 19.05 copies/reaction). The assay showed the same sensitivity in the presence of other substances. Compared to the WOAH-recommended PCR assays, this assay had 10-times higher sensitivity for all tested samples. There were no cross-reactions with other closely related oomycetes, fish muscle, or water samples, indicating that the assay was highly specific for A. invadans. The repeatability and reproducibility tests showed little variation, ranging from 0.1–0.9% and 0.04–1.1%, respectively, indicating the high consistency, repeatability, and reliability of the developed assay. This highly rapid, sensitive, specific, and consistent EUS qPCR assay would be of importance in transboundary disease management and the monitoring of pathogens in aquatic environments. [ABSTRACT FROM AUTHOR]

Despite the growing emphasis on aquatic disease monitoring via environmental DNA (eDNA) in water samples, detecting low concentrations of viruses in aquatic environments remains challenging. Prior studies demonstrated that plankton act as mechanical vectors for several aquatic viral pathogens but not for the viral hemorrhagic septicemia virus (VHSV). In this study, we aimed to identify the potential interaction between VHSV and microplankton and further assess the effectiveness of the water concentration method using a plankton net for VHSV detection in water. Briefly, VHSV was co-cultured with Chlorella , rotifer, and microcosms generated using plankton nets with sieve sizes of 20, 40, and 70 μm. A close interaction between VHSV and rotifer and microplankton collected with the 70 μm-plankton net with an abundant zooplankton population was found, likely influenced by the filter-feeding behaviors of zooplankton. We also found that VHSV could adsorb to fish feces at concentrations 25–88 times higher than that in the equivalent volume of seawater. Subsequently, we evaluated the efficiency of a 70 μm-plankton net for VHSV concentration in rearing water containing infected fish compared to the iron flocculation method. Notably, the plankton net outperformed the iron flocculation method in terms of sensitivity (∼10 to 20-fold), possibly due to its ability to capture fish-derived organic materials, such as feces, shed from infected fish. Our overall findings advocate for the plankton net concentration approach, not only for its pronounced sensitivity but also its operational simplicity, identifying it as a promising avenue for efficient VHSV detection in aquaculture water monitoring. • This study demonstrates an association between VHSV and microplankton. • Concentration of rearing water using a plankton net outperformed iron-flocculation in terms of viral detection. • The plankton net method, with its operational simplicity, can be an effective eDNA tool for monitoring aquatic diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Megalocytiviruses are responsible for severe mortality in various marine and freshwater fish species worldwide. Specifically, the red sea bream iridovirus (RSIV) and infectious spleen and kidney necrosis virus (ISKNV) genotypes are known to cause the red sea bream iridoviral disease (RSIVD). Although several quantitative polymerase chain reaction (qPCR) assays have been developed for diagnosing RSIVD, these methods fail to differentiate between viral genotypes within Megalocytivirus. The present study introduces an innovative multiplex qPCR assay for the precise detection of RSIVD. This diagnostic method targets the histidine triad motif (HIT)-like protein and ORF080L genes, which are unique markers for the RSIV and ISKNV genotypes. The assay exhibited high analytical sensitivity, expressed as the 95% limit of detection (LoD 95%), of 8.69 copies/5 μL for RSIV and 19.07 copies/5 μL for ISKNV. Notably, the assay exhibited no cross-reactivity and was unaffected by common inhibitory substances. The diagnostic sensitivity and specificity were 100% for both RSIV and ISKNV, demonstrating the reliability of the assay. The multiplex qPCR assay was 100–1000 times more sensitive than conventional PCR methods, emphasizing its importance in early-stage infection detection. The repeatability and reproducibility of the assay were confirmed through extensive testing, showing low variation and high correlation across different technicians and laboratory settings. Furthermore, the ability of the assay to selectively detect and differentiate between RSIVD genotypes represents a significant advancement in aquatic animal disease diagnostics. This multiplex qPCR assay offers a rapid, specific, sensitive, and quantitative diagnostic tool for global RSIVD surveillance efforts and contributes to the prevention and understanding of disease progression in the aquaculture industry. • This study presents a multiplex qPCR assay for red sea bream iridoviral disease (RSIVD) detection • The assay detects RSIV and ISKNV, causative agents of RSIVD • The assay showed high analytical performance, with 100% diagnostic sensitivity and specificity for both RSIVD subtypes • This diagnostic tool helps prevent and understand disease progression in the aquaculture industry [ABSTRACT FROM AUTHOR]