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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** 2021 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**
                                

Progress was made on three objectives and their sub-objectives in FY2021, all of which fall under National Program 106, Aquaculture (NP 106) Action Plan Components 1 and 3.

Sub-objective 1a: Third-generation nucleus families from the ARS-Fp/Fc-R (n = 80), ARS-Fc-S (n = 23), and ARS-Fp-R (n = 36) were produced and evaluated for resistance to Flavobacterium columnare. Because upward selection response following two generations of selection for resistance to columnaris disease was less than predicted, the current aim was to evaluate the repeatability of the immersion-based challenge model.  To this end, families were challenged in parallel using the standard, single-family flow-through challenge model and also in a replicated pooled-family water recirculation challenge model.  Resistance to columnaris disease was assessed in both challenge models at approximately 73 days post-hatch, and resistance is currently being assessed in the same families at approximately 130 days post-hatch.  Fin clips were collected from all fish used in the replicated pooled-family water recirculation challenge model and are currently being used to genotype each fish (n = 8,776) to reconstruct pedigrees and obtain family-level survival rates, assess the consistency of family survival rates from each of the three replicates, and to compare family survival between the flow-through and recirculating challenge models.  Similar comparisons will be made following the completion of the 130-day post-hatch challenges, in addition to the assessment of the temporal repeatability of family survival in both challenge models.             

Sub-objective 1b: Rainbow trout from the ARS-Fp-R and ARS-Fp-S genetic lines were injected with saline or live F. psychrophilum, sampled at days 1 and 5 post-challenge, and subjected to whole-organism RNA-sequencing.  This year we identified pathways regulated by the differentially expressed genes between ARS-Fp-R and ARS-Fp-S genetic lines.  ARS-Fp-S line fish had upregulated pathways that included a response to interleukin-1, tumor necrosis factor, and interleukin-6, while ARS-Fp-R line fish exhibited enhanced pathways involved in complement regulation, GTPase activity, neutrophil activity, and metabolism.  To confirm pathway involvement, ten different chemical immune-inhibitors/activators were tested in challenged fish.  One compound abolished ARS-Fp-R line genetic resistance with limited effect on the ARS-Fp-S line.  Additional pathway analyses are ongoing.

Sub-objective 1c: Analysis of available F. psychrophilum genomes identified seven genetic serogoups with a total of 17 probable serotypes.  Progress was made in developing a comprehensive multiplex PCR typing system for rapid molecular characterization of isolates.

Sub-objective 1d: 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.  Laboratory reared fish experienced a water temperature ranging from 12.5C to 13.3C, while fish held in net pens experienced water temperature ranging from 3C to 19.3C.  Spleen samples were collected from both laboratory and farm-reared fish from two genetic lines sampled at 8-time points during grow-out.  Gene expression was measured using RNA-seq, and over 1.35 billion sequences were uniquely mapped to the rainbow trout genome and differentially expressed genes identified.  Many differences were identified between laboratory and farm-reared fish (n=3,036) and between genetic lines (n=777).  Pathways that were upregulated in farmed fish involved the response to type 1 interferon, chemokine activity, response to lipopolysaccharide, and heat stress.  A gene signature of cold adaptation was identified.  Differences between the ARS-Fp-R line and a commercial trout line included the response to heat stress, response to lipopolysaccharide, and serine-type protease activity.  Analysis of gill samples obtained from the fish is ongoing.

Sub-objective 2a: To better understand the role of infection-induced serum factors on virulence regulation in Y. ruckeri we have collected serum from rainbow trout just before and at 3 and 7- days post Y. ruckeri infection. At these times post-infection, we expect a dramatic induction of host antimicrobial factors. These samples are critical for determining the effect of induced serum factors on Rcs system-induced virulence regulation. Experiments examining the effect of Cecropin B on Rcs signaling suggest that this antimicrobial peptide does not affect signaling under the in vitro culture conditions tested. Further work examining the effect of Cecropin B on Rcs signaling when cells are grown in naïve serum will be attempted. In addition, mutation of the Rcs pathway was found to have no effect on the minimal inhibitory concentration of Cecropin B, suggesting that this pathway is not responsible for induced resistance to this antimicrobial peptide.

SubObjective 2b: In collaboration with Dr. Mark McBride, Univ of Milwaukie, 40 mutants of F. columnare have been generated that have targeted gene deletion of genes involved in enzymatic, adhesion, and iron acquisition pathways. These mutants have been tested for virulence in Rainbow trout fry using the waterborne challenge.  Cytolysin A, B, and endonuclease mutants, have reduced virulence.

SubObjective 2c: The addition of supplemental air using diffusers during a F. columnare challenge decreased mortality from 66% to 33%. The dissolved oxygen levels in tanks with supplemented air were >7 mg/L, and in tanks without air diffusers, dissolved oxygen measured approximately 5 mg/L after a one-hour immersion challenge. Further work is currently being conducted to define threshold levels of influence that supplemented air and pure oxygen have during an immersion F. columnare challenge.

Sub-objective 2d: 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 know 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. 

SubObjective 3a: While travel restrictions due to COVID-19 has limited our ability to travel, we have been fortunate that collaborators at Clear Spring Foods (Riverence) have been able to collect and ship samples to the University of Connecticut for analysis. We have established the use of the full-length 16S rRNA gene PacBio sequencing and data analysis. Another collection trip is being planned. After this, our focus will be on characterizing the microbiome and the in vivo biofilms.

SubObjective 3b: Our initial goal was to generate one Flavobacterium columnare and one F. psychrophilum strain that expressed GFP for monitoring biofilm formation. We have been able to establish electroporation to introduce plasmids in F. columnare and were able to introduce a gfp-expressing plasmid into two F. columnare strains belonging to different genomovars. Future experiments will focus on describing the biofilm formation of these strains using confocal microscopy.

Subobjective 3c: We successfully generated and validated a mutant transposon library consisting out of ~40,000 mutants. The library was validated by assessing the frequency of auxotrophs and by Southern blots to ensure the transposons inserted randomly and single insertion are present. Future experiments will focus on performing biofilm experiments and identifying critical genes. **Accomplishments**
                                

1. 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 risk of death over 46-fold and interacted with fish genetic background. This research demonstrated the importance of mitigating effects of poor water quality and improving fish genetics to reduce disease loss. **Review Publications**
                            *Barbier, P., Rochat, T., Mohammed, H.H., Wiens, G.D., Bernardet, J., Halpern, D., Duchaud, E., McBride, M.J. 2020. The type IX secretion system is required for virulence of the fish pathogen Flavobacterium psychrophilum. Applied and Environmental Microbiology. AEM.00799-20. https://doi.org/10.1128/AEM.00799-20.*
                                *Shepherd, B.S., Ma, H., Han, Y., Palti, Y., Gao, G., Liu, S., Wiens, G.D. 2020. Structure and regulation of the NK-lysin (1-4) and NK-lysin like (a and b) antimicrobial genes in rainbow trout (Oncorhynchus mykiss). Developmental and Comparative Immunology. 116 (103961). https://doi.org/10.1016/j.dci.2020.103961.*
                                *Evenhuis, J., Lipscomb, R.S., Birkett, C. 2021. Growth of flavobacterium columnare in TYE with or without supplemented cations. Current Microbiology. 78: 2474–2480. https://doi.org/10.1007/s00284-021-02507-8.*
                                *Evenhuis, J., Lipscomb, R.S., Birkett, C. 2021. Virulence variations of flavobacterium columnare in rainbow trout (Oncorhynchus mykiss) eyed eggs and alevin. Journal of Fish Diseases. 44(5): 533-539. https://doi.org/10.1111/jfd.13343.*
                                *Testerman, T., Beka, L., Mcclure, E.A., Reichley, S., King, S., Welch, T.J., Graf, J. 2021. Detecting flavobacterial fish pathogens in the environment using high-throughput 2 community analysis. Applied and Environmental Microbiology. 6(21):447745. https://doi.org/10.1101/2021.06.21.447745.*
                                *Kloub, L., Gosselin, S., Fullmer, M., Graf, J., Gogarten, J., Bansal, M. 2021. Systematic detection of large-scale multigene horizontal transfer in prokaryotes. Molecular Biology and Evolution. 38(6):2639-2659. https://doi.org/10.1093/molbev/msab043.*


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