Performing Department
(N/A)
Non Technical Summary
Formulated aquafeed has been a primary driver for the growth of the aquaculture industry worldwide. However, assessment of the biosecurity of formulated aquafeed using unvalidated molecular diagnostics (i.e. polymerase chain reaction (PCR)- basedassay poses serious challenges, impacting the ability of shrimp feed manufacturers in the US and elsewhere to address safely and sustainably of the growing domestic and international aquaculture industry due to rejection of consignments.This proposal aims to develop standardized sample handling processes and PCR-based assays curated for testing aquafeed forwhite spot syndrome virus, the most important viral pathogen impacting shrimp aquaculture worldwide, and an emergent fungal pathogen,Enterocytozoon hepatopenaeiinfecting shrimp.The validated assay will be able to differntiate among aquafeed and feed ingredients that may contain fragment WSSV and EHP genome but poses no threat to shrimp health compared to those that may carry infectious pathogens.This will provide a much needed practical solution for the aquafeed industry facilitating the assurance of biosecurity of manufactured feeds until a crustacean cell line is available for screening infectious pathogens in aquafeed.
Animal Health Component
100%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
0%
Goals / Objectives
Manufacturing of formulated aquafeed that serve as an alternative to the use of fresh and unprocessed feeds, known to be of high biosecurity risk, has been a primary driver for the growth of aquaculture industry worldwide. However, the standards in place to determine the biosecurity risk of formulated aquafeeds and feed ingredients have become highly debated in recent years. This is primarily due to the complete dependence on the results of unvalidated PCR-based assay used to detect pathogens in formulated feed and feed ingredient.Historically, PCR-based assays for crustacean diseases were developed using tissue samples from either live experimental shrimp or frozen tissue, and are not curated for a wide array of shrimp feed and feed ingredients such as fish meal, octopus, krill among others that are being tested routinely for a health attestation of aquafeed for international export. Since PCR-based assay cannot differentiate between infectious pathogen and inactivated genomic fragment of the corresponding pathogen, a comprehensive assessment of the biosecurity of aquafeeds using PCR assay is posing a serious challenge directly impacting shrimp feed manufacturers in the US and elsewhere due to rejection of consignments. This scenario is further complicated due to the lack of an immortal cell line in crustaceans. A feed sample tested positive for a shrimp pathogen cannot be evaluated by a cell culture based assay to determine if it contains an infectious pathogen unless a bioassay is performed. Since bioassay and further testing of the experimental animalstakes months, such an approach is not feasible to screen potentially contaminated aquafeed.To overcome this conundrum, we propose to develop a standardized sample handling processes and PCR-based assay curated for testing aquafeed targeting the replicase gene of a pathogen. A basic assumption of this approach is if the replicase gene of a pathogen is fragmented following heat/ other treatment(s) during feed manufacturing, such an aquafeed despite containing fragmented pathogen genome will not cause infection and will pose no biosecurity risk in hatchery/ grow out ponds. We will test this hypothesis by spiking aquafeed with a DNA virus, white spot syndrome virus, and an emergent fungal pathogen, Enterocytozoon hepatopenaei (EHP).Aquafeed ingredients will be spiked with WSSV and EHP inocula and aquafeed will be made in the laboratory following industry standards. Feed will be tested using a validated PCR-based diagnostic assay. Subsequently bioassays will be performed using laboratory made feed and pathogen detected in the experimentally challenged SPF Penaeus vannamei shrimp by histology, in situ hybridizationand PCR targeting the replicase gene. Finally, the optimized PCR assay will be used to detect shrimp pathogens in commercial aquafeed that have been submitted to Aquaculture Pathology Laboratory in recent months and were tested positive by the currently used unvalidated PCR method. The proposed approach is a compromise of the current scenario in crustacean disease detection in aquafeed due to the lack of an immortal cell line. We believe this will be bring an immediate relief to aquafeed manufacturer in the US and make the industry resilient until an immortal cell line is developed for screening live infectious crustacean pathogens in aquafeed.Goal:Develop PCR-based validated assays for attestation of aquafeed for biorisk assessment.Objectives:1. Develop a PCR-based validated assays for screening aquafeed for a viral pathogen, WSSV.2.Develop a PCR-based validated assays for screeningaquafeedfor a fungal pathogen, EHP.
Project Methods
We will develop polymerase chain reaction (PCR)-based mehtods for screening aquafeed and aquafeed ingredients against white spot syndrome virus (WSSV), a major viral pathogen of crustacean and Enterocytozoon hepatopenaei (EHP), an emergent fungal pahogen of shrimp. The PCR assays will be developed targeting the replicase genes of WSSV and EHP. Since a functional replicase gene is a prerequisit for a pathogen to be infectious, if the replicase gene is fragmented during aquafeed manufacturing and thus PCR method fails to amplify this target, the corresponding aquafeed would have no biorisk.We will design several primers to amplify the replicase genes of WSSV and EHP by overlapping conventional PCRWe will also design primers targeting the replicase gene for developing TaqMan real-time PCR assays for the WSSV and EHP.