Progress 09/01/22 to 02/28/25

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?The project provided opportunities for professional development of a researchscientist, Dr. Hung N. Mai working in the Aquaculture Pathology Laboratory, who is also the Co-PI of the project. The PI of theproject, Dr. Arun K. Dhar, the Postdoctoral Fellows and the Co-PI, Dr. Hung Mai had the opportunity to attend national/international conferneces and meetings to share their findings and gain insights of the participants through in-person and in-personinteractions. The project also provided unique opportunitiestoundergraduate students, Tressa M. Baker and M. J. Matthewsworking in Dr. Dhar's lab tolearn disease diagnostics in shrimp using molecular methods. Tressa baker is a author in the manuscript that is currently being considerd for publication. The PI and the Co-PI express sincere gratitude to Dr. Wendy Sealy, Research Scientist,USDA, ARS, Bozeman Fish Technology Center, 4050 Bridger Canyon Road, Bozeman, MT for preparing WSSV and EHP-spikes extruded diet following standard aquafeed industry methods. Producing WSSV- and EHP-spiked feeds were key steps in this project. Dr. Sealy is a co-author in all conference presentation and peer-reviewed publications originating from this project. How have the results been disseminated to communities of interest?The findings of this project werepresented during: 1. The Conference of Research Workers in Animal Diseases, January 22-24, 2024, Chicago, Illinois; 2. Aquaculture America 2024 Conference, World Aquaculture Society,February 18-23, 2024, San Antonio, Texas; 3. USDA Virtual Meeting on Special Research Grants for Aquaculture Research Program, Project Director Meeting, September 04, 2024; 4.Aquaculture America 2025 Conference, World Aquaculture Society, March 06-10, 2025, New Orleans, Louisiana. Peer-reviewed publication: 1. (Based on Objective #1):Mai, Hung N., Schofield, Paul J., Wendy Sealey, Baker, Tressa M. and Dhar, Arun K. 2025. Development of a PCR-based assay for assessing biosecurity risk of aquafeed for white spot disease. Journal of Fish Diseases, Revised manuscript will be submitted soon. 2. (Based on Objective #2):We are currently drafting anothermanuscript based on the data generated for Objective #2. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Goal:Develop PCR-based validated assays for attestation of aquafeed for biorisk assessment. Objective #1: Develop a PCR-based validated assays for screening aquafeed for a viral pathogen, WSSV. Under theobjective #1, we have developed a validated PCR assay for screeningaquafeed and feed ingredients for a viral pathogen of crustaceans, white spot syndrome virus (WSSV). We accomplished this objectives through a series of technical tasks. These include: a)Shrimp diet was prepared followinga standard industry protocol using WSSV-infetced shrimp meal (at 2.0% and 0.2% inclusion rate). A conventional PCR assay targeting the DNA polymerase gene of the virus was developedto screen aquafeed and feed ingredients. The data revealed extrusion process breaks down WSSV DNA to the extent that a feed/ feed ingredient couldbe tested positive by real-time PCR that amplifies ~150 bp amplicon, but tested negative by conventional PCR that generates an amplicon aorund 1000 bp size. Therefore, an optimized conventional PCR amplifying ~1000 bp DNA was developed using the World Organization for Animal Health (WOAH, Paris, France) recommended method.The method could be used for to distinguish infectious WSSV from feed containing non-infectiousgenomic fragments of the viral pathogen. 2. To further prove that extruded diet containing WSSV genomic DNA does not cause white spot disease, experimental diets (at 2.0% and 0.2% inclusion rate) werefed to Specific Pathogen Free (SPF) Penaeus vannamei shrimp andanimals were observed for clinical sign, mortality, and tissues examined by histopathology and conventional PCR assay (see above). 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 spotdisease. 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-spikedmeal, and the virus could not be detected using the PCR assay developed for this project, as well as PCR assay recommended by the WOAH for the detection of WSSV. Objective #2:Develop a PCR-based validated assays for screening aquafeed for a fungal pathogen, EHP. a) Under Objective #2, shrimp diets were prepared following standard industrypractice using shrimp meal (at 2.0% and 0.2% inclusion rate) that was spiked with a known quantity of infectious EHP. Both conventional and real-time PCR assays were developed targeting the DNA polymerase gene of EHP. The data revealed that heating feed ingredients during extrusion process fragments EHP DNA to the extent that a feed/ feed ingredient couldbe tested positive by real-time PCR that amplifies ~150 bp amplicon but tested negative by conventional PCR that generates an amplicon around 1000 bp size. The optimized conventional PCR protocol followed the World Organization for Animal Health (WOAH, Paris, France) method validation steps.The optimized protocol can be used to distinguish infectious EHP from feed containing non-infectiousgenomic fragments of the corresponding parasite (i.e., EHP) DNA. b) To further prove that extruded diet containing EHP genomic DNA does not cause parasitic infection, experimental diets (at 2.0% and 0.2% inclusion rate) werefed to Specific Pathogen Free (SPF) Penaeus vannamei shrimp andanimals were observed for clinical sign and mortality. Additionally, experimental animals were sacrificed and hepatopancreas tissues examined by histopathology and conventional PCR assay (as described under Objective 2a). The results showed that the shrimp diet (containing EHP-infected shrimp meal at 2.0%and 0.2% inclusion rate) prepared following a standard aquafeed industry extrusion method does not cause infection with EHP. None of the experimental animals developed any clinical sign and no mortality was observed throughout the experimental challenge. Additionally, there was no histopathological manifestation in the hepatopancreas, the target organ of EHP in shrimp fed diet containing EHP-spikedmeal, and the parasite could not be detected using the PCR assay developed for this project. Finally, we tested the protocols developed under Objectives #1 and #2 to screen commercial aquafeed and feed ingredients for WSSV and EHP. These aquafeed and feed ingredients were sent to Aquaculture Pathology Laboratory in the University of Arizona for WSSV and EHP screening. Samples tested positive by real-time PCR for WSSV and EHP were found to be negative when the same samples were tested following conventional PCR protocols, as developed under the Objectives 1 and 2. To summarize, we developed PCR-based validated assays that can differentiate infectious WSSV and EHP from genomic fragment of the corresponding pathogens that might be present in formulated feed used to rearshrimp in commerical aquaculture. This will enable to enhance biosecurity of aquafeed in shrimp aquaculture in the US and elsewhere in the world.

Publications

• Type: Other Journal Articles Status: Under Review Year Published: 2025 Citation: Mai, Hung N., Schofield, Paul J., Wendy Sealey, Baker, Tressa M. and Dhar, Arun K. 2025. Development of a PCR-based assay for assessing biosecurity risk of aquafeed for white spot disease. Journal of Fish Diseases (Revised manuscript is being prepared for a submission).
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Hung Mai, Paul Schofield, Wendy Sealey, Arun K. Dhar. DEVELOPMENT OF A PCR-BASED DIAGNOSTIC ASSAY FOR SCREENING Enterocytozoon hepatopanaei (EHP) IN FORMULATED AQUAFEED, Aquaculture America 2025 Conference, World Aquaculture Society, March 06-10, 2025, New Orleans, Louisiana.
• Type: Other Status: Published Year Published: 2024 Citation: Arun K. Dhar and Hung N. Mai. 2024. Development of PCR-based diagnostic assays in support of disease-free attestations of formulated aquafeed. USDA Virtual Meeting on Special Research Grants for Aquaculture Research Program, Project Director Meeting, September 04, 2024
• 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.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Mai, H. N., Schofield, Paul J., Wendy M. Sealey & Dhar, A. K. 2024. Development of a PCR-based diagnostic assay for white spot syndrome virus (WSSV) in formulated aquafeed. The Conference of Research Workers in Animal Diseases, January 22-24, 2024, Chicago, Illinois.

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.

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