Location: Cool and Cold Water Aquaculture Research
2024 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 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 families (n = 27) from the ARS-Fp-S line, were evaluated for resistance to Flavobacterium columnare using a pooled-family water recirculation challenge model, fin clipped, and genotyped to reconstruct pedigrees. Correlation between mid-parent breeding value and mean progeny performance was 0.33, but this correlation estimate may have been adversely impacted by the overall high survival rate for the progeny challenge (88%) compared to mean survival in the parental generation (61%). Body weight at 13 months of age was measured and did not differ among genetic lines (mean = 934 grams; P = 0.35).
Sub-objective 1.b We previously identified infection and vaccination pathways regulated in ARS-Fp-R and ARS-Fp-S genetic lines through whole-organism RNA-sequencing. To further evaluate immune response pathways, we examined the cellular response at day 1 and 5 after bacterial or PBS injection. Bacterial load, plasma biomarkers, blood chemistry and whole blood cellular composition were measured. We examined the effect of an mTOR pathway inhibitor, rapamycin, which results in immunosuppression and a ~100-fold reduction in the challenge LD50. On day 1, bacterial load did not differ between genetic lines and treatment, but by day 5, the R-line exhibited no increase in Fp load, while rapamycin treatment increased ARS-Fp-R and -S line loads 33- and 506-fold, respectively. Biomarkers cathelicidin 2 and C1q-LP3 significantly differed between genetic lines and by time, corresponding to Fp load. Analysis of whole blood by flow cytometry identified a response to infection and a higher frequency of myeloid-gated cells in ARS-Fp-R line fish that was consistent in both naïve and challenged animals. ARS-Fp-S line fish exhibited a higher percentage of IgM+ B-cells. Single-cell RNAseq using purified blood identified 23 cell clusters including multiple B cell, neutrophil and macrophage subpopulations. In naïve animals, S-line fish harbored a cxcr4 and mych expressing B cell subpopulation absent in ARS-Fp-R line fish. Rapamycin pretreatment caused profound loss of multiple B cell subpopulations in ARS-Fp-R and-S line fish. We are currently analyzing multiomic datasets and evaluating sparsity-promoting machine learning algorithms for predictive modeling and biomarker identification.
Sub-objective 1.c: Previously we observed an association of ARS-Fp-R line genetic resistance against F. psycrhophilum strains containing a wzy2 gene and less relative resistance against strains with a wzy1 gene. The wzy2 gene is involved in o-polysaccharide (O-PS) formation, specifically the alpha1-2 linkages between D-Qui2NAc4NR and L-Rha and this difference is associated with the Th serotype. In contrast, the wzy1 gene forms beta 1–3 linkages between D-Qui2NAc4NR and L-Rha in Fp 950106-1/1 and is associated with the Fd 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 Fp CSF259-93 O-PS formed a compact helix whereas the Fp 950106-1/1 O-PS adopted an extended conformation. Using a novel genetic manipulation system, the wzy2 gene in Fp CSF259-93 was replaced with wzy1, and as predicted, mAb FL100A no longer bound bacterial cells. We challenged three genetic lines of rainbow trout (resistant, control and susceptible) with both wildtype Fp CSF259-93 and the wzy2/wzy1-swapped mutant. The relative host resistance of the three genetic lines was not altered when challenged with the wzy2/wzy1-swapped mutant. These results suggest that while the structural variation in the O-PS affects mAb FL100A binding and the humoral response, innate genetic resistance is not impacted by variation in O-PS.
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. We have identified differentially regulated genes in spleen and gill tissue and these data will be combined with plasma proteomic analyses to prioritize candidates for biomarker development.
Sub-objective 2.a: The previously identified Y. ruckeri mutant strain TW32 over-produces flagellin due to a mutation in the promoter region of the flagellar master regulator, flhD. RNAseq analysis performed on this strain suggests that this mutation causes increased expression of FlhD leading to de-repression of the flagellar biosynthesis system and thus unregulated production of flagellin. The RcsB response regulator is a repressor of flagellar biosynthesis that acts by repressing expression of the flhD promoter. It’s possible that RcsB is active in TW32 and that mutational loss of RcsB in the TW32 background could cause further overexpression of flagellin. To test this, we have recently created a rcsB knockout mutant in strain TW32 using marker-exchange mutagenesis. This stain will be used to compare flagellin levels between the single and double mutants by Western blotting.
Sub-objective 2.b: A total of 9 knock-out mutants in Fc exhibited complete or reduced virulence in rainbow trout. Most of these mutants were in components of the type-IX secretion system or gliding motility genes including GldN, PorV, GldJ, SprB, CyclA, CyclB, and multiple secreted peptidases. Mutation of FluA and an ABC transporter associated with iron acquisition and reduced virulence. Surviving fish from the virulence studies were subsequently challenged with the parent strain but no protection was induced by the reduced virulence mutants.
Sub-objective 2.c: Flavobacterium columnare growth and virulence differences were tested in TYES media stored up to 15 days at either room temperature or 4C. Neither factor affected F. columnare growth or virulence, suggesting a minimum TYES media shelf life of 15 days. We also evaluated the effects of air (0.1 cubic meters/minute) and oxygen (0.01 cubic meters/minute) supplementation during F. columnare exposure on pathogen virulence. Whereas air supplementation reduced mortality between 17 and 34% compared to the non-supplemented control group, we observed no effect of oxygen supplementation on pathogen virulence.
Sub-objective 3.a In our screen to identify bacteria that inhibit the growth of Flavobacterium psychrophilum, we identified 24 isolates that could inhibit the growth of F. psychrophilum. Ten of these isolates were selected for genome sequencing. The genomes were analyzed using AntiSMASH to identify genes implicated in antibiotic production. In collaboration with a natural product biochemist, we are working on identifying an active compound from strains that inhibit the growth of F. columnare in a biofilm and F. coave, F. oreochromis, F. psychrophilum, F. branchiophium, Aeromonas salmonicida, Yersinia ruckeri, and K. pneumoniae in coculturing inhibition assays.
Sub-objective 3.b After establishing the biofilm with Aeromonas veronii in the CDC bioreactor, we performed an inSEQ transponson mutagenesis screen. Our initial experiments revealed a strong positive selection for mutants that lost biosynthetic genes for polar flagella, lateral flagella and surface polysaccharides. Increasing the sequencing depth for the 24 h time point allowed us to detect additional genes 24 h after inoculation, which was before the positively selected mutants took over the population cells. We included stainless steel and polycarbonate as surfaces for biofilm formation but detected no differences in inSEQ data. For subsequent experiments, we performed an inSEQ screen in a CDC bioreactor on polycarbonate where F. comlunare formed a biofilm and Aeromonas veronii was added subsequently. inSEQ was performed at the 24 h time to identify mutants that were positively and negatively selected.
Additional: Progress has been made developing tools and methods for the genetic manipulation of W. tructae. Recently we discovered that some strains of W. tructae encode a Type II restriction/modification system with similarity to the HpaII endonuclease. HpaII has a 4bp recognition site that is expected to cut frequently in DNA sequences and likely acts as a strong barrier to transformation. Adapting methods used for the electro-transformation of W. paramesenteroides we have successfully transformed a W. tructae strain lacking this restriction/modification system, thus circumventing this barrier. We have also identified a marker-exchange mutagenesis plasmid, originally designed for use in Staphylococcus aureus and S. epidermidis, that can be used to create mutants in W. tructae. We have demonstrated that this plasmid can transform and replicate in W. tructae and that the resistance marker and counter-selection system in this plasmid are both expressed and functional in this bacterium. We are in the process of validating the utility of this system by creating a marker-exchange mutant strain.
Accomplishments
1. Discovery, validation, and commercialization of a novel diagnostic biomarkers for monitoring salmonid fish health. Fish farmers need rapid methods to assess animal health and wellbeing. ARS researchers at Leetown, West Virginia, St. George's University and a private company identified seven highly regulated proteins present in rainbow trout plasma that correlate with bacterial infection. Quantitative enzyme-linked immunosorbent assays and rapid, no-wash assays were developed. These assays can be completed in 1-2 h time and require only a small amount of blood from fish. The assays are commercially available and provide standardized and rapid methods to monitor rainbow trout and Atlantic salmon population health during grow-out.
2. Disease threat to salmon aquaculture facilities identified. Atlantic menhaden are an abundant migratory marine fish common in large schools along the entire Atlantic Coast. ARS researchers at Leetown, West Virginia, in colloboration with New Jersey Division of Fish and Wildlife collaborators identified serotype 03 Vibrio anguillarum as the agent causing large-scale menhaden kills in the coastal mid-Atlantic region between 2020 and 2021. This pathogen causes significant loss in aquaculture operations world-wide and the serotype 03 strain associated with these menhaden kills is a type that has not been found previously in North America. These results suggest that migrating menhaden could be a potential source for the translocation and transmission of V. anguillarum to salmon aquaculture facilities in the Northeastern US and Canada. This is especially concerning given that V. anguillarum vaccines currently used in these areas are not expected to provide protection against the serotype 03 strain.
Review Publications
Wiens, G.D., Marancik, D.P., Chadwick, C.C., Osbourn, K.E., Reid, R.M., Leeds, T.D. 2023. Plasma proteomic profiling of bacterial cold-water disease resistant and susceptible rainbow trout lines and biomarker discovery. Frontiers in Immunology. 14. https://doi.org/10.3389/fimmu.2023.1265386.
Thunes, N.C., Evenhuis, J., Lipscomb, R.S., Perez-Pascual, D., Stevick, R.J., Birkett, C., Ghigo, J., Mcbride, M.J. 2024. Gliding motility proteins GldJ and SprB contribute to Flavobacterium columnare virulence. Applied and Environmental Microbiology. 206(4). https://doi.org/10.1128/jb.00068-24.
Abraham, T., Yazdi, Z., Littman, E., Shahin, K., Heckman, T.I., Quijano Cardé, E., Nguyen, D., Hua, R., Soto, E., Adkison, M., Veek, T., Mukkatira, K., Richey, C., Kwak, K., Mohammed, H., Ortega, C., Avendaño-Herrera, R., Hyatt, M.W., Keleher, W., Welch, T.J. 2023. Detection Of Lactococcus spp. in environmental samples and wild fish from Four Lakes in Southern California. Journal of Aquatic Animal Health. 35(3):129-198. https://doi.org/10.1002/aah.10188.
Lovy, J., Iwanowicz, L.R., Welch, T.J., Allam, B., Getchell, R., Geraci-Yee, S., Good, C., Snyder, J., Raines, C., Das, N. 2024. Seasonal mortality of Atlantic menhaden is associated with neurologic disease caused by a virulent clone of Vibrio anguillarum: Fish kills and implications for biosecurity. Transboundary and Emerging Diseases. 18:8816604. https://doi.org/10.1155/2024/8816604.
Graf, J., Testerman, T., Varga, J., Donohue, H., Vieira Da Silva, C., Schiffer, M. 2023. Pseudomonas aphyarum sp. nov., Pseudomonas fontis sp. nov., Pseudomonas idahonensis sp. nov. and Pseudomonas rubra sp. nov., isolated from in, and around, a rainbow trout farm. International Journal of Systematic and Evolutionary Microbiology. 73(12):006201. https://doi.org/10.1099/ijsem.0.006201.
Cisar, J.O., Wang, X., Woods, R.J., Cain, K.D., Wiens, G.D. 2024. Structural and genetic basis for the binding of a mouse monoclonal antibody to Flavobacterium psychrophilum lipopolysaccharide. Journal of Fish Diseases. Article e13958. https://doi.org/10.1111/jfd.13958.