Development of a <scp>PCR</scp> ‐Based Assay for Assessing Biosecurity Risk of Extruded Feed for White Spot Disease

Formulated aquafeed has been a primary driver for the growth of the aquaculture industry worldwide. However, formulated feed may pose a biosecurity risk if they are not proven to be free of infectious pathogens. Unforetunately, recent PCR methods cannot differentiate an infectious pathogen from an inactivated genomic fragment of the corresponding pathogen. Considering the lack of an immortal cell line in crustaceans, we developed a conventional one-step PCR assay that can be used to screen formulated aquafeed. As a proof-of-principle, extruded feed containing (w/w) 0.0%, 0.2% and 2% WSSV-infected shrimp meals were prepared and screened using a one-step conventional PCR and a real-time PCR targeting the WSSV DNA polymerase gene. The results were compared to the WOAH-recommended nested- and real-time PCR for WSSV. The newly developed and the WOAH-recommended real-time PCR methods detected WSSV DNA in the extruded feed. Among three primer sets used in one-step conventional PCR, two provided successful WSSV amplification with amplicon size of 334 and 341 bp, respectively. The primer set that generated an amplicon of 980 bp, as well as the WOAH-recommended nested PCR, did not amplify WSSV in the extruded feed. The one-step conventional PCR generating a 980 bp amplicon is highly specific in detecting WSSV, and the limit of detection is 100 copies. When WSSV-spiked extruded feed was fed to Specific Pathogen Free (SPF) Penaeus vannamei shrimp, it did not cause disease, as determined by histopathology and PCR assays, indicating that the WSSV-spiked extruded feed does not contain infectious virus. We propose that real-time PCR, for its high throughput ability, could be used for initial screening, and any samples that test positive for WSSV should be further tested using a one-step conventional PCR that amplifies a 980 bp amplicon. This will provide a much-needed practical solution for biosecurity assessment of formulated aquafeed for WSD until a crustacean cell line is available for screening infectious WSSV.