Progress 07/15/19 to 03/14/20

Outputs
Target Audience:Animal health companies Researchgroups in aquaculture Changes/Problems:The exxpression cassettes tool longer than anticpated to complete and therefore there has been a delay in the resuslts. We do nto anticpate any other changes other than this delay and we plan to complet the goals as outlined n the grant. What opportunities for training and professional development has the project provided?Several undergraduates worked on this project. They were trained in techniques of molecular and cell biology How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Aquaculture is a livestock industry that farms fish for consumption. This growing market is threatened with transmissible diseases that are highly detrimental to growth, but some of these diseases are also transmitted to wild fish.Vaccines are used to protect against disease in aquaculture. However, administration of current vaccines requires individual handling of fish, a difficult and labor-intensive process. Our goal is to develop an effective orally administered vaccine which can be delivered with feed, ensuring that all fish are protected. Pancreatic Disease is one such highly transmissible disease that causes morbidity and mortality in farmed salmon.We have created genes expressing these subnit vaccines and are expressign them in maize grain. This is the first step in the process to be used as the media to deliver the subunti vaccine.This approach would allow a practical solution for the aquaculture industry to delivery efficacious vaccines and control disease. This will lead to more secure supply of this important protein source. The goals and accomplishshments are outlined below. Optimize the codon sequence for antigens E1 and E2 for expression in maize. Codon optimization was completed for both genes. The genes were then synthesized. 2. Design a construct containing both E1 and E2 under the control of an embryo promoter and including a selectable marker. See table below Produce three variations of the construct using different intracellular targeting locations: cell wall, endoplasmic reticulum, and vacuole. See Table below 4. Obtain Agrobacterium transformed with the constructs. Completed 5. Transform maize and obtain at least 10 independent transformants for each construct The following constructs were made transformed in to Agrobacterium and subsequently into maize embryos. Individual transformants were identified, and plants were sent to greenhouse to mature. This goal was accomplished at least for the first 3 constructs. At this time there are still some of the SAE events that may add to the final numbers. Code Candidate Genes Strain Cellular target Marker Gene Events Plants to greenhouse SAB E1 and E2 Strain 3 Cell wall PAT 244 (18 events) 120+ (16 events) SAC E1 and E2 Strain 2 Endoplasmic Reticulum PAT 154 (16 events) 120 (14 events) SAD E1 and E2 Strain 3 Endoplasmic Reticulum PAT 113 (12 events) 80 (10 events) SAE E1 and E2 Strain 2 Cell Wall PAT 27 (4 events) 4 (3 events) 6. Analyze transformants for expression of E1 and E2 using antibodies provided by a commercial partner. We do not yet have the seed from the plants to analyze as the constructs took longer to make than anticipated. We will analyze seed at our expense. 7. Analyze antigens from transformants by gel electrophoresis and western blotting to ensure integrity of the antigens. See above Identify and propagate transformants containing the highest level of expression of the antigens. See above Defat maize flour from identified transformants with supercritical fluid extraction (SFE) to stabilize the antigens. See above 10. Formulate the flour for oral delivery to salmon. See above

Publications