Development of red sea bream iridovirus concentration method in seawater by iron flocculation

Pathogen dynamics in environmental water are considered to be highly associated with the prevalence of fish disease. To examine the dynamics of red sea bream iridovirus (RSIV), which has caused serious damage in marine aquacultures in Japan, we developed a highly sensitive and quantitative method by combining the viral concentration and real-time PCR to detect the virus in seawater. The iron flocculation method employed here successfully concentrated 500 mL of artificial seawater spiked with RSIV in a 0.1-mL DNA solution at a 5000 concentration magnification, and the recovery rate of the RSIV genome by real-time PCR was higher than 80%. The detection limit of this method was approximately 8.0 x 10(1) RSIV copies L-1 of sample water, which is a more than 1000 times improvement compared to the conventional DNA extraction method without concentration of water sample. Using this method, we detected the RSIV genome from the rearing water for experimentally infected Japanese amberjack Seriola quinqueradiata at ranges from 10(2.1) to 10(5.5) copies L-1. The RSIV copy number in the rearing water was increased more than 5 days before mortality started and it might become an indicator of disease prediction. These results indicate that the iron flocculation method coupled with PCR detection is useful for monitoring RSIV dynamics in the aquaculture setting. Statement of relevance: Monitoring of RSIV in environmental water. (C) 2015 Elsevier B.V. All rights reserved.