Location: Cool and Cold Water Aquaculture Research
2025 Annual Report
Objectives
Objective 1. Genetic improvement of rainbow trout for disease resilience.
Sub-objective 1.a Genetic improvement of disease resistance against Fc using the ARS-Fp-R line.
Sub-objective 1.b Identify transcriptional patterns associated with host resistance.
Sub-objective 1.c Define and characterize pathogen determinants influencing host genetic resistance.
Sub-Objective 1.d Measure disease resistance phenotype and performance on-farm.
Objective 2. Improvement of host health through pathogen characterization, vaccine development and characterization of host response to vaccination.
Sub-objective 2.a Molecular-genetic characterization of virulence regulation in Yr mediated by the Rcs pathway.
Sub-objective 2.b Identify virulence factors in Fc by transposon mutagenesis.
Sub-objective 2.c Evaluate environmental factors affecting Fc phenotypes.
Sub-objective 2.d Determine heritability of host response to vaccination.
Objective 3. Identify factors in production system microbiomes that can be used in strategies to improve animal health.
Sub-objective 3.a Determine the microbial composition during biofilm development in raceways.
Sub-objective 3.b Reduce the amount of Fc and Fp in biofilms.
Sub-objective 3.c Evolve Aeromonas to reduce the ability of Fc and Fp to invade biofilms.
Approach
Rainbow trout are a valuable finfish farmed in the U.S. and worldwide. Trout losses from infectious diseases are an important factor limiting production. Three prevalent bacterial diseases of rainbow trout are bacterial cold water disease (BCWD), enteric redmouth disease (ERM), and more recently, columnaris disease (CD). The goals of this project are to 1) develop well-characterized germplasm that exhibits on-farm resistance against multiple bacterial pathogens, 2) determine pathogen virulence mechanisms to aid vaccine development and selective breeding, and 3) characterize and manipulate the microbiome of the aquaculture environment thereby reducing pathogen outbreaks. Our approach incorporates a comprehensive and multidisciplinary strategy that combines selective breeding, quantitative genetics, immunology, and functional genomics of pathogenic bacteria. This research builds on our previous studies in which we developed and released to industry a BCWD resistant line (designated ARS-Fp-R) that has been extensively characterized, and for which we have made progress in uncovering the genetic basis of disease resistance. For the first objective, we continue to improve the ARS-Fp-R line by increasing resistance against CD, determine mechanisms of disease resistance and specificity, and evaluate this line’s on-farm performance in net-pen aquaculture. For the second objective, we characterize virulence factor regulation, evaluate new vaccine candidates for disease prevention and measure the heritability of vaccine response. For the third objective, we utilize metagenomics to define the on-farm microbiome and investigate methods to disrupt pathogen containing biofilms. Results from this research will improve animal well-being, reduce antibiotic use and increase trout production efficiency and profitability.
Progress Report
This is the final report for Project 8082-32000-007-000D which ended December 31, 2024 and summarizes the major accomplishments of the 5-year project. A new approved project 8082-32000-003-00D, entitled “Applying Genetic Improvement, Vaccines, and Biomarkers to Improve Salmonid Fish Health in Aquaculture” has been established. We summarize the major accomplishments by Objective:
Objective 1: Selective breeding improved resistance to bacterial cold-water disease and columnaris disease. Bacterial cold water disease, caused by Flavobacterium psychrophilum, and columnaris disease, caused by Flavobacterium columnare, are important diseases that affect rainbow trout aquaculture. Antibiotics are routinely used to control these diseases because there are limited alternative control strategies currently available. ARS researchers in Leetown, West Virginia, evaluated resistance to both diseases in two unrelated, commercially- relevant rainbow trout nucleus populations. The genetic correlation between resistance to both diseases was found to be favorable in both populations, suggesting that a rainbow trout’s resistance to both diseases is due, at least in part, to a common set of genes. However, four generations of selection for columnaris resistance resulted in only a modest increase in genetic resistance under laboratory conditions. Field studies are required and underway to evaluate disease resistance under natural challenge conditions.
Aquaculture reuse water exposure affects disease susceptibility and survival of farmed rainbow trout. Fish farmers often reuse water to be efficient stewards of freshwater resources; however, reduced water quality is often blamed for disease outbreaks. The magnitude of risk associated with short- or long-term reuse water exposure and its impact on host genetics and vaccine response is unclear. ARS researchers at Leetown, West Virginia, in collaboration with researchers at Virginia Institute of Marine Science and Virginia Tech, varied the duration of reuse water exposure supplied to two commercial strains of rainbow trout that had either been mock-vaccinated or vaccinated against infectious hematopoietic necrosis virus. Chronic reuse water exposure increased the risk of death over 46-fold and interacted with fish genetic background. This research demonstrated the importance of mitigating the effects of poor water quality and improving fish genetics to reduce disease loss.
Discovery, validation, and commercialization of a novel biomarker for susceptibility to bacterial cold-water disease. Fish farmers need rapid methods to assess animal health and disease susceptibility. ARS researchers at Leetown, West Virginia, and St. George's University identified a novel serum biomarker of disease susceptibility.
The biomarker was increased over 20-fold in the plasma of susceptible-line fish following exposure to Flavobacterium psychrophilum. A rapid, no-wash assay was developed and commercialized that can be completed in under 1 h total assay time. This assay provides a commercially available, rapid method for rainbow trout and Atlantic salmon farmers to monitor population health during grow out.
Objective 2: Robust vaccine cross-protection between genetically distinct strains of Weissella tructae (formerly Weissella ceti). Weissellosis, caused by W. tructae, is an economically important emerging disease of farmed rainbow trout that can cause losses as high as 40-80% of production. Until recently, all strains of W. tructae formed a genetically homogeneous group, suggesting a recent emergence of this pathogen. ARS scientists at Leetown, West Virginia, have identified a strain of this pathogen that is genetically distinct from previously characterized strains and likely represents an additional independent emergence of this pathogen. In addition, vaccination experiments established that strong cross-protection is conferred by vaccination by either type indicating that only one strain is needed in the vaccine. This information has been vital to stakeholders for the formulation of a pre- exposure vaccine intended to protect against multiple distinct strains of this pathogen.
Investigation of the heritability of vaccine response. Fifty full-sib families from the ARS-Fp-R line were vaccinated against Lactococcus garvieae (Lg; n = 12 fish per family) or Yersinia ruckeri (Yr; n = 12 fish per family) at approximately 9 months of age, and 84 days later serum samples were collected to evaluate anti Lg-specific and anti Yr-specific IgM antibodies using enzyme-linked immunosorbent assay (ELISA). Whereas individual serum samples were evaluated for Lg antibodies, serum from Yr-vaccinated fish were pooled within a family, and four replicate ELISAs were conducted on each family pool. Endpoint and kinetic ELISA data were recorded for each vaccine treatment and analyzed using a two-trait animal model that included all known pedigree relationships to estimate heritabilities and genetic correlations. Heritability estimates for Lg antibody response were 0 for endpoint and kinetic data, 0.07 for Yr antibody endpoint data, and 0.22 for Yr kinetic data. Genetic correlation estimates of antibody response for both vaccine treatments were 0.02 when assessed as endpoint values and 0.01 when assessed as kinetic values. The low heritability estimates, and lack of genetic correlation suggest that selection for antibody response against these two vaccines in this population will have limited application.
Evaluation of vaccines and water quality parameters on Columnaris disease. Multiple mutations were made in F. columnare that partially or completely impaired virulence for rainbow trout. The mutations altered the function of a type 9 secretion system and gliding motility. Rainbow trout were live-cell vaccinated with mutant strains; however, little protection was observed after challenge with the wild-type parental strain. Several virulence related genes will be expressed as recombinant proteins and evaluated as potential sub-unit vaccines. We found that water hardness and pH influence F. columnare virulence. Calcium and magnesium, two components of water hardness, were necessary for growth and virulence of F. columnare. Water pH modulated laboratory growth in broth media, with a pH approaching 7.0 inducing early onset of stationary growth phase. This resulted in lower overall bacterial densities, leading to less virulence in laboratory challenge.
Objective 3: Detection of fish pathogens by high-throughput analysis. The detection of bacteria that cause disease in fish is critical for the U.S. aquaculture industry to reduce mortality and disease. Diseases caused by Flavobacterium columnare and F. psychrophilum are a major concern because they lead to high mortality and increased use of antibiotics. Researchers at the University of Connecticut developed and validated a high- throughput, next-generation sequencing assay that allows them to detect pathogenic F. columnare and F. psychrophilum in water and on surfaces. The researchers were able to detect F. columnare in water and on the walls of raceways in a commercial trout farm. Using this assay, researchers and diagnostic labs can identify the source of the flavobacterial pathogens and determine when they increase in number. The identification of pathogen refuge and amplification sites will lead to new intervention strategies that improve the quality and safety of the nation’s food supply.
Water source influences the microbiome at a commercial trout aquaculture facility. The microbiome, or collection of microorganisms, can influence development, disease, and overall fish health but remains poorly understood at commercial trout farms. Scientists at the University of Connecticut and ARS Leetown, West Virginia, analyzed 163 microbiome samples collected over a three-year span from fish, water, and tank surfaces at a commercial trout production facility. The incoming water microbiome influences the type of microbes associated with fish and tank surfaces. The fish pathogen Flavobacterium columnare was associated with source water and was prevalent during disease outbreaks. This study identified a potential source and reservoir of an important pathogen and will lead to improved farm biosecurity and disease control.
Accomplishments
Review Publications
Sloboda, S., Ge, X., Jiang, D., Su, L., Wiens, G.D., Beveridge, C.A., Duchaud, E., Mcbride, M.J., Rochat, T., Zhu, Y. 2025. Methylation of foreign DNA overcomes the restriction barrier of Flavobacterium psychrophilum and allows efficient genetic manipulation. Applied and Environmental Microbiology. 91(2). Article e01448-24. https://doi.org/10.1128/aem.01448-24.
Jones, D.R., Everson, J., Leeds, T.D., Wiens, G.D., Wargo, A.R. 2024. Exposure dosage and host genetics influence the shedding kinetics of Flavobacterium psychrophilum in rainbow trout. Journal of Fish Diseases. 48. Article e14026. https://doi.org/10.1111/jfd.14026.
Barcan, A.S., Humble, J., Kasaragod, S., Sajib, M., Barcan, R.A., Mcginnity, P., Welch, T.J., Robertson, B., Vamanu, E., Bacigalupo, A., Llewellyn, M.S., Pedrals, F.S. 2025. Understanding the transfer and persistence of antimicrobial resistance in aquaculture using a model teleost gut system. Animal Microbiome. 7. Article 18. https://doi.org/10.1186/s42523-025-00377-0.