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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** 2023 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:  Family-based selection was practiced on third-generation families and selected broodstock were spawned to produce fourth-generation nucleus families from the ARS-Fp/Fc-R (n = 78), ARS-Fc-S (n = 21), and ARS-Fp-R (n = 43) genetic lines, and ninth-generation nucleus families (n = 27) from the ARS-Fp-S genetic line. These nucleus families are currently being evaluated for resistance to Flavobacterium columnare using a pooled-family water recirculation challenge model (all families from all lines) and the standard single-family flow-through challenge model (17 ARS-Fp/Fc-R and 8 ARS-Fc-S families). Fish from the pooled-family water recirculation Flavobacterium columnare challenge will be fin clipped and genotyped to reconstruct pedigrees, which will enable family-specific survival estimates. These pedigree and survival data will be used in conjunction with similar data from third-generation families to assess accuracy of breeding value estimates and, ultimately, the utility of the pooled-family water recirculation challenge model for improving Flavobacterium columnare resistance in a rainbow trout population through selective breeding.
 
Sub-objective 1b: We analyzed host pathway differences between selectively bred, resistant and susceptible rainbow trout lines. Pooled plasma from unhandled, PBS-injected, and Flavobacterium psychrophilum-injected groups were simultaneously analyzed using a TMT 6-plex label and the relative abundance of 513 proteins were determined and compared to mRNA measured from whole-body RNA-seq. A subset of differentially abundant intracellular proteins was identified, including troponin and myosin, suggesting these proteins were released into plasma following pathogen-induced tissue damage. Validation of disease biomarkers was performed using immunoassays and C1q-LP3, skeletal muscle troponin C, cathelcidin 2, haptoglobin, leptin, and growth and differentiation factor 15 exhibited elevated concentration in susceptible line plasma. Complement factor h like-1 exhibited higher abundance in the resistant-line compared to the susceptible-line in both control and challenged fish and thus was a baseline differentiator between lines.

Sub-objective 1.c:  Previously we 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 (O-PS) formation, specifically the alpha1-2 linkage between D-Qui2NAc4NR and L-Rha and this difference is associated with the Th serotype. This year, we determined that the binding of mouse monoclonal antibody, FL100A, depends on the wzy2-dependent alpha (1-2) linkage and the R group of D-Qui2NAc4NR. In modeling studies, the structure of F. psychrophilum strain CSF259-93 O-PS formed a compact helix whereas the F. psychrophilum strain 950106-1/1 O-PS adopted an extended conformation. These findings identify the monoclonal antibody FL100A as a specific probe for lipopolysaccharide on wzy2 strains of F. psychrophilum. PCR primers were designed against all known F. psychrophilum serogroups and serotypes.  

Sub-objective 1.d: A long-term evaluation of the ARS-Fp-R line performance and survival under farm conditions was conducted and 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. Spleen and gill samples were collected from both laboratory and farm-reared fish from two genetic lines at 8-time points during grow-out and gene expression was measured by RNA-seq. This year, we remapped the sequences to an updated version of the Rainbow trout genome and optimized our bioinformatic pipeline to accurately analyze expression from a problematic subset of tandem, duplicated genes. In addition, we identified sequences from Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease, present in both gill and spleen samples. Parasite sequences were highest at sample time points 1, 5 and 7. The datasets have been deposited in GenBank. 

Sub-objective 2.b: We tested the virulence of 9 mutants associated with a type-IX secretion system and these exhibited either complete or partial virulence attenuation. The genes were either directly involved with the secretion system or gliding motility (GldN, PorV, GldJ and SprB), or associated with the type-IX secretion system (CyclA, CyclB, and 3 peptidases). Also, a mutant with altered FluA and ABC transporter genes, that are involved in iron uptake, had reduced virulence against rainbow trout. All the mutants tested grew at a rate equal to the parent strain, but none conferred protection when surviving rainbow trout were rechallenged with the wildtype strain.

Sub-objective 2c: We evaluated the effect of water hardness on F. columnare virulence. Calcium carbonate greater than 250 mg per L resulted in juvenile rainbow trout mortality of 49% for genomovar I strains, 1% for genomovar II strains, 5% for genomovar IIB strains and 7% for genomovar III strains. Growth of strains in media that lacked magnesium and calcium (components of water hardness) preferentially inhibited growth of genomovar I strains compared to either genomovar II or III strains.

Sub-objective 2.d: No difference in resistance to serum was observed between the Yersinia ruckeri rcsB mutant and its wild-type parent strain. This is consistent with our previous results demonstrating that the Rcs pathway is not induced by serum factors and is not necessary for resistance to antimicrobial factors present in serum. We previously identified a Y. ruckeri mutant strain (TW32) that contains a SNP in the promoter region of the flagellar master regulator causing the production of large amounts of flagellin. Marker-exchange experiments have demonstrated that the SNP in strain TW32 is both sufficient and required for the unregulated flagellin expression seen this strain. 

Sub-objective 3.b: We developed a new slide-based, F. psychrophilum biofilm inhibition assay. Potential inhibitors were allowed to form a biofilm for 48h, after which F. psychrophilum was added and allow to grow six days on the slide. The slides were prepared for fluorescent in situ hybridization and visualized on a confocal microscope. This assay allows comparison to results obtained in co-cultivation experiments on agar plates using a biofilm environment that is more like typical aquaculture settings. We also sequenced 10 bacterial genomes that inhibited the growth of F. columnare and utilized bioinformatic software packages to detect biosynthetic gene clusters involved in the biosynthesis of antibiotics. We are collaborating with a natural products biochemist at the University of Michigan to identify bioactive compounds.

Sub-objective 3.c: We performed four, large scale biofilm growth experiments in a bioreactor with our mutant library of Aeromonas veronii. The biofilms were allowed to form on either stainless steel or polycarbonate. The cells were collected, grown on agar plates and their genomic DNA was extracted and transposon insertion sites sequenced. The power of this approach lies in the ability to identify genes that are positively and negatively selected by determining which genes have either a higher or lower frequency of transposon insertions. In a bioreactor setting, we detected a strong positive selection signal for genes involved in the biosynthesis of flagella and the lipopolysaccharide surface carbohydrate.

Additional work: Disease outbreak investigations at a trout aquaculture facility in Washington State revealed cases of edwardsiellosis (causative agent Edwardsiella piscicida) in fish that had been previously vaccinated against this pathogen, thus suggesting vaccine failure. Genome sequencing and serotyping experiments demonstrated that the strains associated were both genetically distinct and a different serotype then the strain used to produce the vaccine currently in use. A survey of E. piscicida strains isolated at this facility since 2014 showed that this serotype had not been see at the site before the fall of 2022. Future work will focus on defining the role of this novel strain in vaccine failure and developing a more broadly effective vaccination strategy. **Accomplishments**
                                

1. 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. 2. 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 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. **Review Publications**
                            *Graf, J., Testerman, T., Beka, L., Reichley, S.R., King, S., Welch, T.J., Wiens, G.D. 2022. A large-scale, multi-year microbial community survey of a freshwater trout aquaculture facility. FEMS Microbiology Ecology. 98:1-15. https://doi.org/10.1101/2022.05.03.490559.*
                                *Riborg, A., Gulla, S., Colquhoun, D.J., Zeyl Fiskebeck, E., Ryder, D., Verner-Jeffreys, D., Welch, T.J. 2023. Pan-genome survey of the fish pathogen Yersinia ruckeri links accessory- and amplified genes to virulence. PLoS Pathogens. (18)5:e0285257. https://doi.org/10.1371/journal.pone.0285257.*
                                *De Ruyter, T., Littman, E., Yazdi, Z., Adkison, M., Camus, A., Yun, S., Welch, T.J., Keleher, W.R., Soto, E. 2023. Comparative evaluation of booster vaccine efficacy by intracoelomic injection and immersion with a whole-cell killed vaccine against Lactococcus petauri infection in rainbow trout (Oncorhynchus mykiss). Pathogens. (12)5:632. https://doi.org/10.3390/pathogens12050632.*
                                *Thunes, N.C., Mohammed, H.H., Evenhuis, J., Lipscomb, R.S., Perez-Pascual, D., Stevick, R.J., Birkett, C., Conrad, R.A., Ghigo, J., Mcbride, M.J. 2023. Secreted peptidases contribute to virulence of fish pathogen Flavobacterium columnare. Frontiers in Cellular and Infection Microbiology. 13. Article:1093393. https://doi.org/10.3389/fcimb.2023.1093393.*
                                *Vallejo, R.L., Evenhuis, J., Cheng, H., Fragomeni, B.O., Gao, G., Liu, S., Long, R., Shewbridge, K., Silva, R.O., Wiens, G.D., Leeds, T.D., Martin, K.E., Palti, Y. 2022. Genome-wide mapping of quantitative trait loci that can be used in marker-assisted selection for resistance to bacterial cold water disease in two commercial rainbow trout breeding populations. Aquaculture. 560(738574). https://doi.org/10.1016/j.aquaculture.2022.738574.*


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