Optimization of iron flocculation for enrichment of CyHV-2 in aquaculture water

Cyprinid herpesvirus 2 (CyHV-2) is an important infectious virus endangering the farmed Carassius species industry. The enrichment and identification of CyHV-2 in the water is a vital means of early warning diagnostics, which is helpful to aquatic diseases prevention and control. In this study, we employed and optimized iron flocculation to enrich the concentrations of CyHV-2 in aquaculture water. Different Fe 3+ concentrations (0.5, 1.0, and 10.0 mg/L), various membrane filters (mixed cellulose ester, glass fiber, nylon microporous filter membrane), different elution buffers containing reductants (ascorbate, citrate, DL-malate, and their combinations with EDTA or TE buffer) were compared to check the recovery yield of CyHV-2. Viral genome quantitation was determined using quantitative real-time PCR. The results showed that Fe 3+ concentration of 1.0 mg/L, the glass fiber and citrate-TE were determined to have high efficiency and stable recovery yield. To evaluate infectious CyHV-2 viral titres, crucian carps ( Carassius auratus ) were intraperitoneally injected with CyHV-2. The mortality rate was 100 % at 9 days postinoculation (dpi) with the 96.7 % detection of CyHV-2 in the pooled tissues. The histopathological phenotype showed necrotic cell death in the liver, spleen and kidney. The CyHV-2 genome was ranging from 10 1.1 to 10 7.5 copies/μl in the deceased fish, and 10 1.4 to 10 3.0 copies/μl in the feeding water at various stages from latent infection to onset of disease. The field samples from Huzhou, Zhejiang Province on June 4th and 7th had higher detection rates, 87.5 % and 100 % respectively, corresponding to higher CyHV-2 detected in the aquaculture water. CyHV-2 viral load in cultured waters tends to increase prior to fish death, which provides a new indicator for the prediction of fish diseases. In summary, we have established a unified operational standard for enrichment of CyHV-2 in the aqueous environment by iron flocculation. • Established an iron flocculation process for CyHV-2 enrichment in aquaculture water. • Determined the optimal Fe 3+ concentration to be 1.0 mg/L. • Screened the glass fiber with high filtration efficiency. • Screened the high efficient citrate-TE buffer with convenient storage. • Successfully applied iron flocculation to monitor the dynamic changes of CyHV-2 in aquaculture water.