Progress 09/01/23 to 08/31/24
Outputs Target Audience:• Aquafeed industry in the US and elsewhere in the world. • Researchers and professionals from academia, industry, government, and non-governmental organizations working on aquafeed and animal nutrition, shrimp and fish diseases, and aquatic animal health. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The findings of this project was presented during the Aquaculture America 2024 Conference, World Aquaculture Society, February 18-23, 2024, San Antonio, Texas. What do you plan to do during the next reporting period to accomplish the goals? We will continue analyzing the EHP spiked feed and optimize the PCR assay so that the assay could distinguish infectious EHP from non-infectious EHP (i.e. genomic fragment of EHP that might be present in formulated aquafeed). Feed healthy shrimp with shrimp diet containing EHP-spiked shrimp meal (at 2.0% and 0.2% inclusion rate) and determine if the diet causes infection in healthy shrimp. Based on the findings, develop an optimized PCR protocol to screen aquafeed and feed ingredients for EHP.
Impacts What was accomplished under these goals?
Objective #1 has been accomplished. Under this objective, we have developed a validated PCR assay for screening aquafeed and feed ingredients for a viral pathogen of crustaceans, white spot syndrome virus (WSSV). Shrimp diet was prepared for a standard industry protocol using shrimp meal (at 2.0% and 0.2% inclusion rate) that was spiked with known quantity of infectious WSSV. A PCR assay targeting the DNA polymerase gene of the virus was developed to screen aquafeed and feed ingredients. The method could distinguish infectious WSSV from feed containing non-infectious genomic fragments of the viral pathogen. Then, experimental diet (as described in #a) was fed to Specific Pathogen Free (SPF) Penaeus vannamei shrimp and animals were observed for clinical sign, mortality, and tissues examined by histopathology and PCR assay developed to screen aquafeed and feed ingredients. The results showed that shrimp diet (containing WSSV-infected shrimp meal at 2.0% and 0.2% inclusion rate) prepared following a standard aquafeed industry extrusion method does not cause white spot disease. There was no clinical sign of white spot diseases and no mortality in the experimentally challenged animals. Additionally, there was no histopathological manifestation in the WSSV target organs in shrimp fed diet containing WSSV-spiked meal, and the virus could not be detected using the PCR assay developed for this project as well as PCR assay recommended by the World Organization for Animal Health (WOAH, Paris, France). The results showed extrusion method of preparing shrimp diet inactivates infectious WSSV and the PCR assay developed for this project could differentiate infectious WSSV from non-infectious viral genomic fragments that might be present in aquafeed. We are working to complete the tasks under Objective #2. Shrimp diets were prepared following standard industry practice using shrimp meal (at 2.0% and 0.2% inclusion rate) that was spiked with a known quantity of infectious EHP. A PCR assay targeting the DNA polymerase gene of EHP has been developed. We are now optimizing the method to screen aquafeed to determine if it the newly developed PCR assay could distinguish infectious EHP from feed containing non-infectious genomic fragments of EHP.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Arun K. Dhar, Hung N. Mai, Paul J. Schofield, & Wendy M. Sealey. 2024. Overcoming challenges in disease free certification of aquafeed and feed ingredients by Polymerase Chain Reaction (PCR)-based diagnostic assay. Aquaculture America 2024 Conference, World Aquaculture Society, February 18-23, 2024, San Antonio, Texas.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Hung N. Mai, Paul J. Schofield, Wendy M. Sealey and Arun K. Dhar. 2024. Development of a PCR-based diagnostic assay for white spot syndrome virus (WSSV) in formulated aquafeed. Aquaculture America 2024 Conference, World Aquaculture Society, February 18-23, 2024, San Antonio, Texas.
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Progress 09/01/22 to 08/31/23
Outputs Target Audience:• Aquafeed industry in the US and elsewhere in the world. • Researchers and professionals from academia, industry, government, and non-governmental organizations working on aquafeed and animal nutrition, shrimp and fish diseases, and aquatic animal health. Changes/Problems: Delay in receiving Specific Pathogen Free (SPF) juvenile shrimp (~15 g size animals) from Florida. FedEX lost a shipment of juvenile shrimp due to bad weather early this year. This set us back in starting our live animal experiments since we needed large size animals for generating plenty of virus -infected biomass for making formulated feed. Lack of personnel for the delay in hiring process led to a shortage of researchers in the lab for accomplishing the technical tasks on time. For these reasons, we asked for a No Cost Extension, and it was approved. What opportunities for training and professional development has the project provided?
Nothing Reported
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? We will be analyzing the WSSV spiked feed and challenged animals by utilizing newly developed primers as well as OIE-recommended primers. We will also generate EHP spiked aquafeed and optimize the PCR assay for EHP detection. Lastly, we plan to conduct an EHP challenge test similar to the one we conducted with WSSV spiked aquafeed.
Impacts What was accomplished under these goals?
Objective#1: Formulation of shrimp feed spiked with infectious WSSV and EHP Major activities completed / experiments conducted A bioassay to generate WSSV-infected tissues was conducted. The WSSV-infected tissues were then used to produce aquafeed. Data collected WSSV-infected tissue was generated with the load of 3.07 x 107 copies/mg of tissue Two aquafeeds containing 2 loads of WSSV were produced. Summary statistics and discussion of results The WSSV spiked feed was used to feed the shrimp in the bioassay to see if the WSSV spiked feed could give the infection to the challenged shrimp. The WSSV spiked feed, and the negative control feed were used to optimize the PCR protocol to detect WSSV in the feed. Key outcomes or other accomplishments realized. WSSV spiked feed was used as a material to develop PCR protocols that do not give false positive results. Objective#2: Develop optimized PCR protocols for WSSV and EHP detection in formulated aquafeed. Major activities completed / experiments conducted DNA extraction method for aquafeed was determined. New primers for WSSV and EHP detection in aquafeed were developed. PCR optimization for newly developed primers was conducted. Data collected Four DNA extraction methods showed the same efficacy in DNA extraction. New primers for WSSV and EHP were designed based on polymerase-encoded genes. Regarding specificity, the new primer sets detected only WSSV and EHP. Regarding sensitivity, the limit of detection (LOD) of new assays was 10-100 copies per reaction. The diagnostic sensitivity and specificity of WSSV-PCR were higher than 90%. Summary statistics and discussion of results Four DNA extraction methods can be used interchangeably in DNA extraction from feed samples. The specificity and sensitivity of the new protocol were in acceptable range in diagnostic. Key outcomes or other accomplishments realized. RSC-Maxwell 16, one of tested DNA extraction methods was an automatic extraction platform. The utilization of this platform in DNA extraction from feed will save time and reduce the returning time of the testing. Newly developed primers should run side-by-side with the OIE-recommended primers to evaluate the efficacy of new primers in diagnostic sensitivity and specificity determination. Objective#3: Validation of newly developed PCR assay to assess biosecurity risk of formulated experimental feeds containing heat-inactivated WSSV and EHP Major activities completed / experiments conducted A bioassay using WSSV spiked feed was conducted. Data collected Shrimp fed with WSSV spiked feed showed no mortality meanwhile the positive control tank showed 100% mortality.
Publications
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