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 Cool and Cold Water Aquaculture Research: Leetown, WV
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ARS Home Northeast Area Leetown, West Virginia Cool and Cold Water Aquaculture Research Research





- Wiens, Gregory - Greg
- Welch, Timothy - Tim
- Evenhuis, Jason
- Leeds, Timothy - Tim
- 2023
- 2022
- 2021
- 2020
- Publications
- Aquaculture (106)

Research Project: Improving Salmonid Health through Breeding, Vaccination and Microbiome Modulation **Location:
                            Cool and Cold Water Aquaculture Research** 2022 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**
                                

Sub-objective 1.a: Genotyping of all third-generation families from the ARS-Fp/Fc-R, ARS-Fp-R, and ARS-Fc-S lines to reconstruct pedigrees from the pooled-family water recirculation columnaris challenge model was completed. Mean family survival was 41.6%, 38.5%, and 33.4% for the three lines, respectively, when challenged at 73 days post-hatch. Twenty five nucleus families were also challenged using our original flow-through columnaris challenge model. There was little correspondence of family survival between both contemporaneous challenges (correlation = 0.39), which suggests the presence of unknown non-genetic effects affecting consistency across our challenge models. Our efforts to assess repeatability of the pooled-family water recirculation columnaris challenge were unsuccessful as low mortality (< 1%) was observed when the fish were challenged at 130 days post-hatch. As second-generation nucleus families were evaluated for resistance to columnaris disease in the flow-through challenge model, the small correlation between mid-parent estimated breeding values and progeny survival (r = 0.20) and resulting lack of selection response was not unexpected. Third-generation families from all three lines have been evaluated for growth performance to market weight. Family-based selection will be practiced to produce fourth-generations families, and these families will be evaluated in the pooled-family water recirculation challenge model to assess the utility of this new challenge model to selectively breed for improved resistance to columnaris disease.   

Sub-objective 1b: Rainbow trout from the ARS-Fp-R and ARS-Fp-S genetic lines were sampled at days 1 and 5 post-challenge with live F. psychrophilum and subjected to whole-organism RNA-sequencing. We examined pathways regulated between days 1 and 5 post-challenge. Bacterial load increased in susceptible-line fish between days 1 and 5 while conversely, bacterial load decreased in resistant-line fish. Pathways associated with increased inflammation were identified in ARS-Fp-S line fish while pathways involved in wound repair, complement regulation, and metabolism were identified in ARS-Fp-R line fish. To confirm pathway involvement, immune-inhibitors were tested in both time-course and dose-response experiments and by single-cell sequencing of peripheral blood leukocytes isolated from control and treated fish.  

Sub-objective 1.c: We previously observed an association of ARS-Fp-R line genetic resistance against F. psycrhophilum strains containing a wzy2 gene. The wzy2 gene is involved in o-polysaccharide formation, specifically the alpha1-2 linkage between D-Qui2NAc4NR and L-Rha which defines an antigenic serotype. In collaboration with scientists at Minnesota State University, the wzy2 gene from Fp strain CSF259-93 was swapped with the wzy1 gene of strain 950106-1/1. The wzy-swapped strain exhibited reduced virulence in both ARS-Fp-R and ARS-Fp-S line fish.  

Sub-objective 1.d: As part of the long-term evaluation of the ARS-Fp-R line performance and survival under farm conditions, we compared gene expression between fish reared in the laboratory at our center (constant environment) to fish reared in net-pens located on the Columbia River. Gill samples were collected from both laboratory and farm-reared fish from two genetic lines at 8-time points during grow-out. Gene expression was measured using RNA-seq, and over 1.13 billion sequences were uniquely mapped to the rainbow trout genome and differentially expressed genes identified.  Farm-reared fish gill tissue exhibited upregulation of genes involved in heat-shock response, B-cell infiltration, and inflammation.  

Sub-objective 1.a: Competitive co-culture growth assays were used to compare the ability of the Yersinia ruckeri rcsB mutant to compete with its wild-type parent strain when grown in serum from naïve fish and serum collected 3 or 7 days after challenge. These experiments demonstrated that the rcsB mutation did not cause a fitness disadvantage when grown in serum collected either before or after challenge, suggesting that the Yr Rcs pathway is not required for resistance to serum factors. In addition, growth in serum did not cause the repression of flagellin seen previously during infection. This demonstrates that the Rcs pathway is not activated by infection-induced serum factors and suggest that additional host signals may be responsible for regulation.

Sub-objective 2.b: Over 200 F. columnare mutants have been generated and tested for virulence. Most mutants associated with the type-nine secretion pathway have partial to complete virulence attenuation. 

Sub-objective 2.c: Dissolved calcium and magnesium affect water hardness and we demonstrated the concentration of these elements influences growth of F. columnare in broth culture. Reduction of calcium and/or magnesium inhibited genomovar I, F. columnare growth but not other genomovar types. Genomovar I, F. columnare exclusively causes outbreaks of Columnaris disease in rainbow trout aquaculture and monitoring or modulating water chemistry may be useful for reducing loses associated with F. columnare.

Sub-objective 3.a: We established the use of a bioreactor for the biofilm assays instead of a flow cell as this allows us to determine more parameters of biofilm formation. GFP-expressing Flavobacterium columnare allowed the quantification of biofilms using confocal microscopy.

Sub-objective 3.b: A total of 511 bacterial isolates were cultured from an Idaho trout farm and the associated water source using a co-culture system. The ability of isolates to inhibit the growth of F. columnare, F. psychrophilum, Aeromonas salmonicia, and Yersinia ruckeri were determined using plate culture. Thirty-three isolates inhibited at least one of the fish pathogens and two inhibited the growth of F. columnare. The 33 strains were further characterized by testing against a total of 5 different F. columnare strains and 24 isolates inhibited one or more F. columnare strains. In an independent assay, these strains were tested with a GFP-expressing F. columnare strain that formed a biofilm in multi-well plates and the biofilm formation was assessed by quantifying the GFP signal.

Sub-objective 3.c: A bioreactor was used to grow replicate biofilms to screen a transposon (Tn) mutant library, and we demonstrated that we can sample over time for up to 96 h. This setup allows us to grow the biofilms on strainless steel and polycarbonate surfaces and recover the transposon mutants that formed a biofilm on these surfaces. We established a bioinformatic workflow to identify the transposon insertion sites using data generated from A. veronii grown in a rich and defined growth medium. We sequenced ~800,000 Tn insertions in each of five samples, mapped them to the genome and calculated the Tn insertions per insertion site for each of the 4,256 genes that could potentially harbor the transposon.  

Additional: Disease outbreak investigations at a trout farm in North Carolina revealed the first cases of Weissellosis (causative agent Weissella ceti) in vaccinated fish since vaccination for this pathogen started in 2014. Genome sequencing of the strains recovered demonstrated a mutation in a gene predicted to function the in biosynthesis of a surface antigen. Antigenic analysis of these strains confirmed antigen loss in this strain. Future work will investigate the possibility that this change in antigenicity allows these variant strains to subvert the immunity elicited by the commercial vaccine thus causing vaccine failure.

Additional: In collaboration with scientists from the New Jersey Division of Fish and Wildlife we identified and characterized two pathogens associated with a large-scale disease event in Atlantic menhaden that occurred along the mid-Atlantic coast in 2020 and 2021. The pathogens identified, Vibrio anguillarum and Yersinia ruckeri, both cause serious disease problems in cultured salmonid fish. The V. anguillarum strains identified are serotype 03 which has not been reported previously in North America. Migration of menhaden along the eastern Atlantic could provide a potential vector for the spread of this V. anguillarum strain to Atlantic salmon aquaculture facilities in the Northeastern U.S. **Accomplishments**
                                

1. 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 the 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. 2. Early life-stage model of Columnaris disease developed. Rainbow trout susceptibility to Columnaris disease, caused by Flavobacterium columnare, is an emerging problem in the U.S. aquaculture industry. ARS researchers at Leetown, West Virginia, developed a pre and post hatch life-stage, disease challenge model and found that host genetic background, pathogen strain and water quality altered disease onset and mortality. This model system allows for large scale evaluations to elucidate virulence mechanisms and screen vaccine candidates that can be used to reduce Columnaris disease on farm.
. 3. 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 farmers of rainbow trout and Atlantic salmon to monitor population health during grow-out. **Review Publications**
                            *Nguyen, D., López-Porras, A., Marancik, D., Hawkins, L., Welch, T.J., Petty, B., Ware, C., Griffin, M.J., Soto, E. 2021. Genetic characterization of heterologous Edwardsiella piscicida isolates from diverse fish hosts and virulence assessment in a Chinook salmon Oncorhynchus tshawytscha model. Journal of Fish Diseases. 44(12):1959-1970. https://doi.org/10.1111/jfd.13509.*
                                *Shahin, K., Veek, T., Heckman, T.I., Littman, E., Mukkatira, K., Adkinson, M., Welch, T.J., Imai, D.M., Pastenkos, G., Camus, A., Soto, E. 2021. Isolation and characterization of Lactococcus garvieae from rainbow trout, Onchorhyncus mykiss, from California, USA. Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.14250.*
                                *Riborg, A., Gulla, S., Strand, D., Wiik-Nielsen, J., Ronneseth, A., Welch, T.J., Spilsberg, B., Colquhoun, D.J. 2022. qPCR screening for Yersinia ruckeri clonal complex 1 against a background of putatively avirulent strains in Norwegian aquaculture. Journal of Fish Diseases. https://doi.org/10.1111/jfd.13656.*
                                *Hansen, J.D., Ray, K., Chen, P., Yun, S., Elliott, D., Conway, C., Calcutt, M., Purcell, M., Welch, T.J., Soto, E. 2021. Disruption of the Francisella noatunensis orientalis pdpA gene results in virulence attenuation and protection in zebrafish. Infection and Immunity. 89(11):220-21. https://doi.org/10.1128/.*
                                *Thunes, N.C., Conrad, R.A., Mohammed, H.H., Zhu, Y., Barbier, P., Evenhuis, J., Perez-Pascual, D., Ghigo, J., Lipscomb, R.S., Schneider, J., Li, N., Erbes, D.H., Birkett, C.L., LaFrentz, B.R., Welch, T.J., McBride, M.J. 2021. Type IX secretion system effectors and virulence of the model Flavobacterium columnare strain MS-FC-4. Applied and Environmental Microbiology. 88(3). Article e01705-21. https://doi.org/10.1128/aem.01705-21.*
                                *Everson, J.L., Jones, D., Taylor, A., Rutan, B., Leeds, T.D., Langwig, K., Wargo, A.R., Wiens, G.D. 2021. Aquaculture reuse water, genetic line and vaccination affect Rainbow trout (Oncorhynchus mykiss) disease susceptibility and infection dynamics. Frontiers in Immunology. 12: Article 721048. https://doi.org/10.3389/fimmu.2021.721048.*
                                *Vallejo, R.L., Cheng, H., Fragomeni, B.O., Silva, R.O., Martin, K.E., Evenhuis, J., Wiens, G.D., Leeds, T.D., Palti, Y. 2021. The accuracy of genomic predictions for bacterial cold water disease resistance remains higher than the pedigree-based model one generation after model training in a commercial rainbow trout breeding population. Aquaculture. 545:737164. https://doi.org/10.1016/j.aquaculture.2021.737164.*


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