Location: Cool and Cold Water Aquaculture Research
2020 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 FY2020, all of which fall under National Program 106, Aquaculture (NP 106) Action Plan Components 1 and 3: Problem Statement 1B. Define Phenotypes and Develop Genetic Improvement Programs; Problem Statement 3A. Improve Understanding of Host Immunity, Immune System Evasion by Pathogens, and Disease-Resistant Phenotypes, and Problem Statement 3B. Control of Pathogens and Prevention of Disease.
Sub-objective 1.a: Fish from 99 ARS-Fp/Fc-R, 35 ARS-Fp-R, 23 ARS-Fc-S, 27 ARS-Fp-C, and 33 ARS-Fp-S nucleus families were evaluated for growth performance through 13 months post-hatch. A total of 40 ARS-Fp/Fc-R and all 23 ARS-Fc-S, as well as fish from the ARS-Fp-R, ARS-Fp-C, and ARS-Fp-S mixed-family pools, were selected and retained as broodstock for spawning in January 2021 to produce third-generation families divergently selected for Flavobacterium columnare resistance and eighth-generation families from the Flavobacterium psychrophilum resistance selective breeding program.
Sub-objective 1.b: Whole fish from the ARS-Fp-R and ARS-Fp-S genetic lines of rainbow trout that had been injected with four treatments and sampled at two time points were subjected to RNA-seq. Sequence reads were aligned to the rainbow trout genome and the number of sequences aligning to 55709 gene features were counted. Eight-five percent of the protein coding genes were expressed. Differences in gene expression between genetic lines and treatments were identified by sixteen pair-wise comparisons calculated using the program DESeq2. Thousands of differentially expressed genes were detected and the treatment-activated gene expression pathways are being determined.
Sub-objective 1.c: LPS was purified from Flavobacterium psychrophilum strains ARS-060-14 and CSF117-10. These two isolates differ antigenically and are predicted to differ in R-groups attached to the sugar Qui2NAc4NR. The LPS was cleaved to release the O-polysaccharide and was subjected to glycosyl composition analysis by gas chromatography-mass spectrometry.
Sub-objective 1.d: As part of a long-term evaluation of ARS-Fp-R line performance and survival in net pens located on the Columbia river, spleen samples were collected from ARS-Fp-R line fish and a commercial control-line reared either in on-farm net pens or under defined laboratory conditions. Total RNA was extracted from 340 spleen samples. RNA has been pooled and subjected to RNA-seq to identify genes that are differentially expressed between environments.
Sub-objective 2.a: The micro micro-chemostat system has been designed and constructed and conditions for continuous culture at low temperature have been determined. This equipment will be critical for assessing the role of serum factors in RcsB signaling.
Sub-objective 2.c: The addition of calcium carbonate and/or magnesium sulfate to the TYES growth media (normal growth media) resulted in Flavobacterium columnare isolates growing at normal or expected rates. Tyrptone and yeast extract media without added calcium carbonate and/or magnesium sulfate interfered with growth of genomovar I isolates but had little effect on the growth rates of genomovar II or III isolates.
Sub-objective 2.d: Antibody titers were determined using ELISA for approximately 660 ARS-Fp/Fc-R fish each from three vaccine treatments (Lactococcus garvieae, Yersinia ruckeri, and mock vaccine). Titer data will be analyzed using an animal model to determine the heritability of host response to vaccination.
Sub-objective 3.a: The goal of this sub-objective was to establish the technology to follow biofilm composition over time using a combination of 16S rRNA deep sequencing and fluorescent in situ hybridization (FISH) imaging of biofilms. We obtained probes that target eubacteria, CFB bacteria, Proteobacteria of the Alpha 4 group, Rhodobacterales, Acinetobacter, Comamonas, Flavobacterium, and Aeromonas. We tested the probes against pure cultures and biofilms but the optimization and design of additional probes was delayed by stoppage of laboratory work.
Sub-objective 3.b: We exceeded the original milestone of identifying 100 bacterial isolates cultured from raceways of a commercial trout farm. To date, we were able to identify 438 isolates belonging to the phyla Actinobacteria, Bacteroidetes, Deinococcus, Firmicutes, and Proteobacteria that comprised 22 Families and 45 Genera. Of these isolates 35 were able to inhibit the growth of at least one other bacterium during our initial screen where we tested for inhibition of Aeromonas salmonicida, Yersinia ruckeri, Flavobacterium psychrophilum and F. columnare. Most of the isolates that inhibited the growth of other bacteria were Pseudomonas spp, followed by Flavobacterium spp. The isolates were also tested for biofilm inhibition.
Sub-objective 3.c: The goal of this sub-objective was to validate an INSeq mutagenesis approach in Aeromonas veronii. We have obtained a transposon with the appropriate antibiotic resistance markers and have been able to introduce it into Aeromonas using conjugation. The approach was validation by Southern blots and quantifying the frequency of auxotrophic mutants.
Accomplishments
1. Newly-identified bacteriophage with unique activities promises to prevent disease in rainbow trout. Bacteriophage (or phage) are viruses that infect and kill bacteria and, in the process, can amplify themselves tremendously. Used against disease-causing microbes, these self-replicating killers are excellent candidates for the prevention or treatment of bacterial diseases and could be safe and sustainable alternatives to traditional antibiotics. ARS researchers at Leetown, West Virginia, have identified a new bacteriophage that kills the rainbow trout pathogen Yersinia ruckeri. This phage is unique because, in addition to killing its bacterial host by infection, it also binds to and degrades lipopolysaccharide, a large carbohydrate structure that covers the surface of some bacteria and provides protection from the trout immune system. By trimming off this protective layer the phage renders Yersinia ruckeri susceptible to the trout immune system making it unable to survive inside its fish host. This phage therefore has two independent ways to kill bacteria giving it an antimicrobial one-two punch.
Review Publications
Good, C., Davidson, J., Straus, D.L., Harper, S.B., Marancik, D., Welch, T.J., Peterson, B.C., Pedersen, L., Lepine, C., Redman, N., Meinelt, T., Liu, D., Summerfelt, S. 2020. Assessing peracetic acid for controlling post-vaccination Saprolegnia spp.-associated mortality in juvenile Atlantic salmon Salmo salar in freshwater recirculation aquaculture systems. Aquaculture Research. 00:1-4. https://doi.org/10.1111/are.14567.
Gulla, S., Bayliss, S., Björnsdóttir, B., Dalsgaard, I., Haenen, O., Jansson, E., Mccarthy, U., Scholz, F., Vercauteren, M., Verner-Jeffreys, D., Welch, T.J., Wiklund, T., Colquhoun, D.J. 2019. Biogeography of the fish pathogen Aeromonas salmonicida inferred by vapA genotyping. FEMS Microbiology Letters. 366(7). https://doi.org/10.1093/femsle/fnz074.
Welch, T.J. 2019. Characterization of a novel Yersinia ruckeri serotype O1-specific bacteriophage with virulence neutralizing activity. Journal of Fish Diseases. 43(2):285-293. https://doi.org/10.1111/jfd.13124.
Birkett, C., Lipscomb, R.S., Moreland, T.D., Leeds, T.D., Evenhuis, J. 2020. Recirculation versus Flow-through Rainbow Trout Laboratory Flavobacterium Columnare Challenge. Diseases of Aquatic Organisms. 139: 213–221. https://doi.org/10.3354/dao03487.
