Location: Dairy Forage Research2011 Annual Report
1a. Objectives (from AD-416)
The Great Lakes region is home to approximately 32% of the U.S. population which consumes more than 1.5 billion pounds (valued at $23 billion, 2004 dollars) of seafood products annually. Additionally, the Great Lakes sports and commercial fisheries are estimated to generate $4.5-$5.0 billion and $1 billion per year, respectively. To maintain these fisheries, as well as access to fresh seafood (finfish) products in this region, stocking efforts rely heavily upon regional aquaculture producers, as well as producers who service the fresh seafood (finfish) markets. Yellow perch are a high-value food fish ($10.00-$15.00/lb retail) and current ARS (and ARS partners) efforts are aimed at developing improved yellow perch germplasm and rearing/production practices to advance aquaculture in this region. The recent emergence of viral hemorrhagic septicemia (VHS) virus in the Great Lakes region of North America has led to dramatic multi-species epidemics (affecting 28 Great Lakes fish species). This disease poses a serious threat to yellow perch (aquaculture) production and profitability in this region, and thus, scientific efforts of this ARS program. Consequently, additional work is needed to ensure the cost-effective production of pathogen-free stocks of cultured yellow perch. To address these needs, we shall undertake the following objectives for this period of the cooperative agreement: 1) Generate F3 progeny and evaluate genetic and environmental sources of variation in growth (performance trials) and susceptibility to the VHS pathogen, 2) Continue development of cost-effective early feeding methods to improve yellow perch fingerling survival and growth, 3) Continue to characterize innate immune response and early pathogen recognition pathway(s) to VHS (and subunits), in vitro (from cell lines) and in vivo (VHS challenge studies), and 4) Validate and optimize a new internal standard mixture for the STaRT-PCR assay for rapid detection of VHS.
1b. Approach (from AD-416)
1. Initiate genotype x environment (GxE ) studies on F3 progeny to evaluate growth and susceptibility to the viral hemorrhagic septicemia (VHS) pathogen. F2 broodstocks will be crossed and progeny reared to approximately 5 g. Approximately 10,000 fingerlings/strain will be sent to a commercial facility to evaluate performance (growth and survival), and an additional 2,000-3,000 fingerlings/strain will be sent to the Western Fisheries Research Center to undergo VHS challenge studies. Fingerlings will also be provided to other Great Lakes WATER Institute (GLWI) researchers for other on-site performance trials. Depending on the outcome of the crosses, a larger performance trial may be initiated for F3 progeny that will extend for 12 months. Tissue samples will be collected from animals in these studies for eventual genotyping. For animals exposed to the VHS pathogen, additional tissue samples will be collected and processed for RNA to analyze gene expression using a quantitative polymerase chain reaction assay and/or microarrays. Processed tissues will also support other objectives to develop a rapid polymerase chain reaction (PCR)-based (STaRT-PCR) method to detect the VHS pathogen. 2. Investigate the intensive aquaculture technology (IAT) of perch fingerling production. We will continue to investigate the early-life stage feeding protocols for yellow perch. The primary goals of this research are to reduce dependence on live feeds, improve survival and growth, and reduce fingerling production cost. Live food from the onset of first feeding will be shifted to commercial diets. To improve growth and survival, we will also modify environmental rearing conditions such as photoperiod, food presentation, stocking density, temperature, etc. 3. Validate (biological and technical) the STaRT-polymerase chain reaction (PCR) assay using a new standardized mixture of internal standards (SMIS). We will determine the lower limit of detection and quantification, accuracy, precision, and linear dynamic range of the modified SMIS. Internal standards for ACTB, EF1alpha, 18S rRNA and VHS are included in this SMIS and will be validated. When technically validated, the STaRT-PCR assay will be biologically validated. Specifically, changes in viral load over time will be determined, as well as differences in viral particle number between immersion and injection challenges and between fish with and without clinical signs of hemorrhagic disease. We will also generate viral stocks of known titer, as well as isolates from putative natural VHS outbreaks, for further testing and verification of assay parameters. 4. Continue dissecting functions of VHS structural and non-structural genes in perturbing cellular innate immune responses. We will assess individual VHS proteins in viral replication, innate immune gene induction, IFN–dependent viral inhibition, and innate immune signaling pathways in fish cells. This work will include a wide array of cell-based studies aimed at dissecting the roles of VHS P, M and NV genes in viral replication and dissemination.
3. Progress Report
Three early life stage trials with yellow perch were conducted. Density was adjusted relative to eggs, sac-fry, larvae & post-larvae in flow-through systems. The baseline trial started with 15 egg ribbons per tank, but fingerling production was low, 4500 fish per tank. Subsequent trials reduced the number of egg ribbons; using only 9 egg ribbons, fingerling production increased to 35,000 fish per tank. Evaluation of genetically-defined yellow perch broodstocks continued. Full & half-sib crosses have been completed on F2 Choptank & Perquimans River broodstock strains to produce F3 fingerlings. Performance trials are underway using flow-through & static systems & also at two commercial aquaculture producers. Animals in the top 40% in terms of growth performance from F2 Perquimans, Choptank & Winnebago strains were genotyped to ~90%. Efficiency of genotyping assays has been improved via multiplex PCR assays for microsatellite analysis. Pathogen challenges studies, using the viral hemorrhagic septicemia (VHS) virus, were performed in collaboration with USGS. In addition, experiments were performed by USGS to determine if survivors of VHS challenges could carry VHS virus & subsequently infect naive fish. F3 Perquimans, Choptank & Winnebago strains were sent to USGS for a cross strain challenge to determine if there is genetic variation for VHSv susceptibility in genetically-defined yellow perch. Fish cell lines were developed to characterize viral recognition & response in fish. To accomplish this, the major VHSv IVb proteins (M, P, G, N and NV) were cloned into eukaryotic expression vectors for expression in fish cells. Expression plasmids for fish interferon (IFN) & fish mitochondrial antiviral signaling gene (MAVS) were acquired to study their roles in cellular detection & resistance to the VHSv pathogen. Both M & P proteins inhibited cellular antiviral responses, while NV appeared to augment innate immune response. In viral replication studies, co-expression of the VHSv M or P proteins enhanced viral cytopathic effects & led to decreased IFN production by cells while NV expression decreased viral cytopathicity. Both IFN & MAVS conferred upon fish cells absolute resistance to VHSv. Pretreatment of cells with IFN fully blocked VHSv replication, but infection of cells 24 hours prior to treatment with IFN overcame its ability to prevent VHSv replication or cell death. New oligonucleotide primer sets were designed to amplify additional housekeeping genes for control & standardization of the StaRT-PCR assay. Also, new primer sets were designed to amplify all VHSv strains (I-IV) to improve detection of the virus. The VHSv primer sets were tested for specificity against various human & fish viruses as well as various VHSv isolates. The VHSv-all strains primer set amplified only the VHSv isolates. Specificity & sensitivity experiments were conducted along with optimization of strand-specific reverse transcription and PCR conditions. Experiments thus have verified the analytical characteristics of the StaRT-PCR test. ADODR monitoring is accomplished through on-site meetings, conference calls, individual phone calls and e-mails with cooperators.