Source: VIRGINIA INSTITUTE OF MARINE SCIENCE (VIMS) submitted to
RETHINKING SELECTIVE BREEDING FOR HEALTH: REDUCED PATHOGEN SHEDDING AS A NOVEL COMPONENT OF MULTI-TRAIT ENHANCEMENT IN SALMONID AQUACULTURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1030637
Grant No.
2023-70007-40203
Cumulative Award Amt.
$300,000.00
Proposal No.
2022-06040
Multistate No.
(N/A)
Project Start Date
Jul 1, 2023
Project End Date
Jun 30, 2025
Grant Year
2023
Program Code
[AQUA]- Aquaculture Research
Project Director
Wargo, A. R.
Recipient Organization
VIRGINIA INSTITUTE OF MARINE SCIENCE (VIMS)
COLLEGE OF WILLIAM AND MARY
GLOUCESTER POINT,VA 23062-1346
Performing Department
(N/A)
Non Technical Summary
Infectious hematopoietic necrosis virus (IHNV) is a widespread problem for the rainbow trout (Oncorhynchus mykiss) aquaculture industry, resulting in large scale production losses and economic hardship. Selective breeding of fish for resistance to IHNV offers a powerful option for management of this devastating pathogen and mitigation of its impacts. In particular, selective breeding for fish that are resistant to infection and transmission is likely to be the most effective strategy, because it has the potential to facilitate virus eradication from production facilities. In this project, we plan to quantify variation in IHNV transmission resistance across rainbow trout genetic lineages (families) and estimate the heritability of the trait. We will the determine if transmission resistance is linked to other farm performance traits such as resistance to clinical disease, growth, yield, and fillet quality. This will lay the foundation for future campaigns to selective breed rainbow trout for IHNV transmission resistance. The project will involve a partnership between a leading expert in IHNV biology, Andrew Wargo, PhD at the Virginia Institute of Marine Science, and the Chief Science Officer, Jesse Trushenski, PhD at a large rainbow trout aquaculture company, Riverence Holdings. Results will be disseminated across the salmonid aquaculture and disease management communities through presentations at professional associations, industry forums, and scientific meetings; as well as through publications in trade and scientific journals for dissemination to the larger community. Collectively, this project will not only bolster IHNV management in salmonid aquaculture, but provide a framework for selective breeding of transmission resistance that could be applied to a variety of aquaculture systems.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3114030110150%
3033711108150%
Knowledge Area
303 - Genetic Improvement of Animals; 311 - Animal Diseases;

Subject Of Investigation
3711 - Trout; 4030 - Viruses;

Field Of Science
1101 - Virology; 1081 - Breeding;
Goals / Objectives
The goal of the proposed work is to identify a new collection of broad-based traits that can be used in conjunction to breed healthier, more robust livestock and enhance the long-range genetic potential of intensively cultured salmonids. Our long-term goal is to develop a selection regimen that produces stocks with high resilience and vigor that are genetically equipped to thrive. In particular, we will focus on elucidating if IHNV transmission resistance (e.g., reduced viral shedding) is linked with clinical disease (e.g. morbidity and mortality) resistance as well as other performance traits. We focus primarily on IHNV resistance because it remains one of the most problematic infectious disease issues in salmonid aquaculture and we believe large gains can be made by rethinking IHNV management and refocusing genetic improvement strategies on IHNV transmission resistance and multi-trait selection [53]. We will achieve this through a partnership between Riverence Holdings LLC and the Virginia Institute of Marine Science (VIMS), leading institutions in aquaculture, aquatic health, virology, and selective breeding.
Project Methods
Obj. 1: Quantify disease resistance of trout families against infectious hematopoietic necrosis virusApproximately 80-100 trout families will be produced by single dam/sire pairings in each year of the project in the course of Riverence's ongoing selective breeding program. Families will be hatched and reared in segregation until they reach 2 months of age, in a biosecure facility. Juvenile fish will then undergo a standard IHNV virulence challenge using established protocols at Riverence. This will involve exposing duplicate tanks of 50 fish per family to a controlled dosage of IHNV (locally isolated strain; 2 x 105 pfu/ml) through static immersion in water for 1 hour. Fish will be reared according to routine practices and mortality will be tracked for 21 days. Survival analysis will be conducted to elucidate differences in IHNV disease resistance between families. These studies will be repeated in year 1 and 2 of the project, to allow for investigation of a larger number of trout families and increase replication. Combining this with data from previous generations of broodstock at Riverence, including molecular pedigrees and relatedness, will enable us to estimate heritability for IHNV disease resistance.Obj. 2: Quantify IHNV transmission resistance capacity of trout familiesTo get an initial assessment of viral shedding at a family level, we will sample water from all tanks in the family-based IHNV challenge studies (Obj. 1) at two time points (2-4 days post-challenge) to capture the peak in viral shedding. We will then process samples for IHNV using quantitative PCR to get a preliminary assessment of which families differ in shedding. A subset of 50 trout families (~20 from a IHNV resistance-selected line, ~20 from an unselected line, and ~10 resistance-selected × unselected crosses to generate families of half-siblings to the other families) undergoing virulence challenges in Obj. 1 will then be randomly selected for further IHNV transmission resistance quantification, using established protocols. Groups of 20 fish per family will be exposed to a controlled dosage of IHNV (same locally isolated strain used by Riverence; 2 x 105 pfu/ml) in a 1-hr static immersion challenge. Each fish will then be separated into individual 0.8L tanks on a tower rack system (Aquaneering) to track viral shedding at individual fish level using quantitative RT-PCR. The fish will be held in static flow for three days with aeration (previous experience indicates water quality is sufficiently maintained when applying this protocol), at which time a water sample will be taken from each tank and processed via quantitative RT-qPCR for IHNV quantification. As for Obj. 1, heritability of transmission resistance will be estimated by comparing variation among families with estimates of relatedness generated as part of Riverence's ongoing selective breeding practices. Further, heritability estimates will be determined on an individual basis by comparisons between full- and half-siblings and unrelated individuals (i.e., if transmission resistance is heritable, shedding rates will be most similar between full-siblings, least similar between unrelated individuals, and intermediate between half-siblings).Obj. 3: Correlate IHNV transmission and disease resistance traits with other performance traits of trout families and on-farm performance in a commercial settingIn addition to its ongoing IHNV viral challenge testing, Riverence has developed a series of indicators of biological fitness related to growth performance and efficiency, health, stress tolerance. Families are assessed relative to these traits in the context of 'sentinel' experiments conducted annually. These are essentially common garden experiments in which representative numbers of individuals from each family group are commingled and reared together according to standard commercial practices. The performance data gathered from the sentinel experiments will be evaluated in conjunction with the IHNV resistance and shedding data described in the preceding objectives to determine whether correlations exist among these traits. Groups of eggs are collected from each of the 50-100 family pairings used in Objs. 1-2 and incubated such that all groups hatch at the same time (eggs that are spawned earlier are reared in colder water to extend the incubation time as needed). After hatch, all groups are commingled and reared as a production lot according to standard commercial practices. Once the fish reach market size, representative individuals are assessed regarding the performance metrics/traits of interest and genotyped to reassign individuals back to family groups. In this manner, families can be evaluated in terms of unbiased, 'real-world' performance and these data can be compared with the aforementioned IHNV resistance and shedding data generated for the same family groups.Obj. 4: Develop combined, multi-trait, performance metric for trout selective breeding to improve fish health outcomesWe will combine data from various performance-based metrics (disease resistance - Obj. 1, transmission resistance - Obj. 2, sentinel field performance - Obj. 3) to develop a selective breeding strategy for higher performance families across multiple traits. The first step of this process will involve determining the positive or negative associations between traits. We will construct appropriate linear models to elucidate overall correlations between various traits across families. Heritability estimates will be calculated for the various metrics based on each family's performance and estimates of relatedness among the families. The possible coheritability of breeding targets, including IHNV disease resistance and transmission resistance, will be examined further by assessing whether these traits co-occur in individuals identified as over-performing ('winners') and under-performers ('losers') in the sentinel experiments. Briefly, as the average size of the sentinel population reaches the desired market weight (e.g., ~500 g or 1.25 lbs), all fish will be graded to determine the approximate size distribution of the population. Those individuals in the top 5-10% will be considered winners, those in the bottom 5-10% will be considered losers. Winners and losers will be evaluated according to Riverence's comprehensive phenotypic assessment, including various morphometric and physiological measurements. Additionally, winners and losers will be subjected to genetic pedigree analysis to determine their family of origin, allowing end-of-trial sentinel metrics (i.e., the aforementioned sentinel phenotypic assessment) to be associated with pre-trial metrics (i.e., results of the IHNV challenges and other biological fitness assessments conducted as part of the Riverence breeding program) and the creation of comprehensive profiles describing the combinations of phenotypes exhibited by over- and underperforming individuals. Although the number of individuals sampled is generally large (1000-2000 individuals are assessed at the end of each sentinel experiment), it is unlikely that all families will be represented among the winners and losers. Nonetheless, the results of this analysis will allow for real-world ground-truthing of coheritability among traits identified by the simple correlation analysis. The benefit to our approach is that it allows us to identify individual families which perform well in multiple traits as targets for future selection. This will allow us to develop an overall scoring system for each family across multiple-traits. We intend to apply this methodology after year 1 of the project to inform breeding for year 2. This will allow us to provide initial estimates of heritability of various traits, in particular, transmission resistance, which has not been previously investigated in this system.

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:Our target audience for year 1 of the project was students, laboratory technicians, the scientific community, disease managers, aquaculturists, and government agencies. Our student audience was focused on training in microbiology and immunology skills, such as virology, experimental design and setup, sample collection, sample processing, and pathogen detection using quantitative PCR. Laboratory technician audiences were also trained in similar skills. We disseminated our results at 3 scientific meetings, reaching scientists, disease managers, and aquaculturists. The goal of these interactions was to inform the conference attendee audiences about the application of selective breeding for transmission resistance as a tool for disease management in aquaculture. We also engaged in regular monthly communication with industry partners at Riverence Holdings to share data and provide guidance on their selective breeding program. Our government agency interactions were with the USDA National Center for Cool and Cold Water Aquaculture in Leetown West Virginia. We shared our results with USDS NCCCWA researchers and discussed collaborative projects for using our samples and data to extend our proposed work and investigate genetic markers for transmission resistance in fish. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?At lead institution VIMS we have trained one faculty member, one laboratory technician, one undergraduate student, and one graduate student. The faculty member gained experience in project management, budget management, experimental oversight, personnel mentoring, and results dissemination. The laboratory technician gained training in experimental management, independently conducting experiments, sample processing using molecular techniques, data collation, and supervision of students. Specialized training in new digital droplet PCR technologies to quantify viral shedding was also provided to the lab technician for this project. The undergraduate and graduate student both gained experience in assisting with experimental setup, sample collection, and sample processing. All of these skills are pertinent to careers in animal health and disease management, particularly in the field of virology. At partner institution Riverence we trained one Chief Science Officer, one Brood Manager, and one lab assistant. The Chief Science officer gained experience in sub-award oversight and management, experimental oversight at sub-award institution, personnel management, and dissemination. The Brood Manager gained experience in experiment management, incorporating transmission resistance into selective breeding, and data collation. The lab assistant gained experience in experiment setup and sampling support, collation of samples, and development of experimental protocols. How have the results been disseminated to communities of interest??To date we have disseminated our results at 3 scientific meetings: Ecology and Evolution of Infectious Disease Collaborative meeting in London, UK; Ecological Society of America Annual Meeting in Long Beach, CA; and American Fisheries Association Annual Meeting in Honolulu, HI. At each of these meetings we presented our results on the linkage between IHNV transmission and disease resistance in rainbow trout and their benefits as targets for selective breeding. Attendees at these meetings included academic scientists, government researchers, veterinarians, aquaculturists, fish disease managers, and aquaculture technology support industry representatives. Lead institution VIMS and sub-awardee industry partner Riverence also engaged in regular communication (monthly-bimonthly) to discuss project planning and results. This resulted in the integration of viral shedding resistance data to inform the rainbow trout selective breeding program at Riverence. Lastly, dissemination was achieved through educational materials for "Ethics in Epidemiology" and "Fundamentals of Aquatic Health" classes taught by PI Wargo at VIMS. These materials addressed best practices for the usage of selective breeding as a tool for pathogen management in agriculture. What do you plan to do during the next reporting period to accomplish the goals?For the next project period, we will conduct another set of large-scale studies. To complete the year 1 studies, we will integrate other farm performance traits, (yield, growth, fillet quality, on farm survival) into our selective breeding matrix, in addition to IHNV disease and transmission resistance, for our year 1 brood year fish. We will also conduct additional experiments in year 2. One study will involve the characterization of approximately 200 rainbow trout families for resistance against disease caused by IHNV, at Riverence holdings. This will in essence be a repeat of experiment 2 in year 1, but with the use of a new brood year of rainbow trout that has undergone one generation of selective breeding with transmission resistance as a selection target. This will allow us to determine if additional gains in performance through inclusion of our new shedding resistance metric. We will also evaluate other farm performance traits for these families at Riverence. Furthermore, we will also conduct an additional shedding experiment in year 2, to characterize the transmission resistance of approximately 30 families of brood year 2 rainbow trout. We will expand these studies beyond those conducted in year 1 experiments, by exposing the fish to a range of IHNV dosages. This will allow us to calculate parameters such as the dosage of virus needed to infect 50% of fish as well as better characterize variation in shedding resistance within families. This will provide more powerful metrics for elucidating the IHNV resistance of individual families and allow for more informed estimation of the heritability of these traits. We will also preserve fish from these studies for potential future expansion of this work to identify the genetic markers associated with IHNV transmission resistance in rainbow trout, with our collaborators at USDA NCCCWA. We will continue our dissemination efforts with stakeholders. We aim to submit 1-2 manuscripts for publication in year 2 of the project. We will also present our results at 2-3 scientific meetings. We will continue our discussions with industry partners at Riverence and their associated collaborators on best practices for selective breeding. We will continue to train the project personnel described in year 1 throughout year 2 of the project in important aquatic health skills. We anticipate training and additional 1-2 students in year 2 of the project, beyond those trained in year 1.

Impacts
What was accomplished under these goals? We completed two large scale experiments in year of the project. For experiment 1, we evaluated the IHNV transmission resistance of approximately 30 rainbow trout families, at the Virginia Institute of Marine Science. This was achieved by conducting viral shedding experiments, where fish were exposed to a controlled dosage of IHNV and then peak viral shedding was measured via quantitative PCR. Viral shedding was used as a proxy for transmission. We found that there was significant variation in shedding, and as such transmission resistance, between families, suggesting that it is a genetically linked trait. For experiment 2, we evaluated the resistance of approximately 200 rainbow trout families against disease caused by IHNV. These studies were conducted at Riverence holdings and involved exposing groups of fish to a standard dosage of IHNV and then monitoring mortality over the course of infection. We found significant variation in disease resistance across rainbow trout families. The rainbow trout families in experiment 1 were also included in experiment 2, which allowed us to investigate the correlation between shedding (i.e. transmission) resistance and disease resistance in rainbow trout. We found that there was a strong positive correlation, with families for resistant to disease also typically being more resistant to transmission. However, there were notable exceptions with some highly disease resistant families still having high shedding loads, and thus high transmission potential. These year 1 results were used to inform selective breeding of rainbow trout families at Riverence for production and use in year 2 experiments. Breeding is focused on families with both high disease and transmission resistance. Fish for these year two studies have already been spawned and hatched and are awaiting growth to appropriate size before conducting experiments. An additional extension of the year 1 studies was a collaboration developed with USDA NCCCWA to investigate genetic markers of IHNV transmission resistance in rainbow trout, using full genome sequencing and transcriptomics. Fish from experiment 1 in year 1 of the project have been preserved for these studies. We have already developed a new paradigm of incorporating transmission resistance into selective breeding, which is the major goal of our project.

Publications