Location: Cool and Cold Water Aquaculture Research
2017 Annual Report
Objectives
Objective 1: Define phenotypes and identify genetic markers to enhance selective breeding for disease resistance.
Sub-objective 1.a. Selective breeding for improved CD resistance using the ARS-Fp-R line.
Sub-objective 1.b. Evaluate approaches to exploit intra-family genetic variation for disease resistance to BCWD.
Sub-objective 1.c. Fine-map the Omy19 BCWD QTL and determine mechanism of increased survival.
Sub-objective 1.d. Evaluate survival, performance, environmental effects, and IHNV vaccination of ARS-Fp-R line in a 2015 large-scale field trial.
Sub-objective 1.e. Evaluate ARS-Fp/Fc-R line in field trials.
Sub-objective 1.f. Develop Fp and Fc isolate databases and elucidate genomic and virulence variation.
Objective 2: Improve vaccine development through pathogen characterization.
Sub-objective 2.a. Characterize expression of the Yr flagellar secretion phenotype during the infection process and characterize the role of flhDC in flagellar secretion regulation.
Sub-objective 2.b. Identify flagellar regulatory elements and identify the flagellar secretion component(s) which antagonize virulence in Yr.
Sub-objective 2.c. Evaluate strain TW32 as a live attenuated vaccine strain and as a novel carrier vaccine for en masse delivery of protein antigens to fish.
Sub-objective 2.d. Delineate the molecular, structural and antigenic repertoire of the O-polysaccharides(O-PS) present in Fp and develop typing reagents.
Objective 3: Genomic characterization of bacterial-host-environmental interactions leading to the disease state.
Sub-objective 3.a. Metagenomic analysis of the aquaculture environment.
Sub-objective 3.b. Determine the importance of Type III Secretion systems in mesophilic Aeromonads causing disease in rainbow trout.
Approach
Rainbow trout are a valuable finfish farmed in the U.S. and worldwide. Loss of trout from infectious disease is an important factor limiting production efficiency. Three prevalent bacterial diseases of rainbow trout are bacterial cold water disease (BCWD), enteric redmouth disease (ERM), and recently emerging, columnaris disease (CD). The goals of this project are to 1) develop well-characterized germplasm that exhibits dual on-farm resistance to both BCWD and CD, 2) utilize pathogen genomics to aid vaccine development and selective breeding, and 3)characterize both the host and aquaculture microbiome(s) associated with pathogen outbreaks. Our approach incorporates a comprehensive and multidisciplinary strategy that combines selective breeding, quantitative genetics, immunophenotyping, 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 (ARS-Fp-R) that has been extensively immunophenotyped, and have made progress in uncovering the genetic basis of disease resistance. In the first objective, we initiate selective breeding to improve CD survival, evaluate on-farm performance of single and double pathogen resistant lines and identify strategies for improving selective breeding for disease resistance. In the second objective, we characterize virulence factor regulation, develop serotyping tools, and evaluate new vaccine strategies to prevent disease. In the third objective, we utilize metagenomics and functional-genetic analyses to define the microbiome, identify virulence factors, and elucidate the contribution of these factors to disease outbreaks. The overall impact of this research is improved animal well-being, reduced antibiotic use and increased production efficiency.
Progress Report
Progress was made on all three objectives and their sub-objectives in FY2017, 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.
Columnaris disease, which is caused by Flavobacterium columnare, is an emerging problem in rainbow trout aquaculture, and we previously demonstrated that resistance to this bacteria is a heritable trait that can be improved by selective breeding. Under Objective 1.a, divergent selection was practiced in a pedigreed population (ARS-Fp-R), which was previously subjected to five generations of selection for improved resistance to bacterial cold water disease (BCWD), for resistance to columnaris disease, with the aim of developing double-resistant (ARS-Fp/Fc-R), control (no selection; ARS-Fp-R), and susceptible (ARS-Fc-S) lines of rainbow trout. After one generation of selection, survival of ARS-Fp/Fc-R families (n = 100) was 2.5 percentage points higher, and ARS-Fc-S families (n = 23) 16.6 percentage points lower, than families (n = 33) from the ARS-Fp-R line when subjected to a controlled laboratory challenge with a single isolate of F. columnare. The asymmetric selection response was unexpected and is currently unresolved, and will be the subject of future investigation. Pedigreed fish from all lines and families were retained for subsequent breeding to produce second-generations families in 2019.
Under Objective 1.c, we made progress toward understanding the genetic basis of resistance to bacterial cold water disease by further mapping a quantitative trait locus (QTL) on rainbow trout chromosome Omy19. In collaboration with ARS research project project 8082-31000-012, a combination of single nucleotide polymorphism (SNP) genotyping and RAD sequencing approaches were used to analyze fish previously phenotyped for BCWD resistance. Analyses confirmed a QTL on Omy19 segregating in families 2012474 and 2012473 but not in families 2012287 and 2012288. A putative recombination event was identified on the end of chromosome Omy19 that presumably explained the difference between families. New SNPs have been identified and are being validated to improve the localization of the Omy19 BCWD QTL using a custom Fluidigm genotyping assay. The homeologous regions between Omy19p and Omy10q have made the precise mapping of the QTL challenging. Using pedigreed offspring from families 2012473 and 2012474, a total of 22 additional families were generated in 2017. These families were challenged with F. psychrophilum at 2 gram body weight and fin tissue samples were collected from each family. DNA extraction and genotyping efforts are underway. Candidate genes within the QTL region are being evaluated. In collaboration with scientists at Middle Tennessee State University, over 500 non-coding RNA’s were identified that have different levels of abundance among three genetic lines of rainbow trout that vary in disease resistance. Their identification revealed immune and physiologic mechanisms associated with disease resistance and provide new targets for measuring the immune response.
Under Objective 1.d, a 2015 production-scale farm trial was completed at a commercial farm site in Idaho and performance results reported in last year’s annual report. This year we completed biochemical measurement of plasma samples collected during this trial from fish reared either at the National Center for Cool and Cold Water Aquaculture (NCCCWA) or the farm site at two time points: approximately 2500 and 5200 temperature degree days. Parameters measured included total protein, albumin, globulin, glucose, calcium, cholesterol, sodium, potassium, chloride and packed cell volume. Differences in plasma parameters were observed both between sites and time points indicating age and environmental influence on plasma biochemistry profiles measured in ARS-Fp-R line fish.
Under Objective 1.e, a large-scale farm-trial was initiated with a commercial producer and trial design was modified based on stakeholder input and facility availability. The trial is being conducted on the Columbia River where significant loss of large-sized rainbow trout during summer months have been attributed to columnaris disease. The trial will compare the survival of a production lot of triploid, ARS-Fp-R line fish with the survival of an age-matched triploid commercial line. The disease resistant phenotypes of both lines were measured at the NCCCWA using standardized BCWD and columnaris challenge procedures. Fish are scheduled for net-pen delivery in August 2017 and performance data will be collected through harvest, estimated to occur in early 2019.
Under Objective 1.f, we determined and published the genome sequence of isolate CSF-298-10. A total of 2,911 genes and 318 subsystems were identified. This strain is currently being used to select a line of rainbow trout with enhanced columnaris disease resistance. In addition, 24 F. columnare genomes belonging to 4 different genomovars have been recently draft sequenced and assembled. Preliminary analyses using whole genome phylogenetic comparison are consistent with substantial genetic differences between genomovars. Within-genomovar groups were similar in sequence and exhibited greater than 95% nucleotide identity.
Under Objective 2, we fulfilled a component of sub-objective 2.b by identifying the rcsB gene as the flagellar regulatory element which controls virulence in Y. ruckeri. Mutation of rcsB resulted in unregulated expression of flagellin during infection and concomitant attenuation of virulence, likely due to flagellin-mediated host immune stimulation. A number of additional genes that are regulated by rcsb were identified and their role in virulence will be determined. Mutants lacking the fliC or ylpA genes have been constructed in the TW32 genetic background for future studies contributing to sub-objective 2.b. In addition, a gfp fusion construct has been developed and moved to appropriate strains for future vaccine delivery studies under Objective 2.c.
Under Objective 3.a, we surveyed the microbial community to determine whether sequences belonging to Flavobacterium columnare or Flavobacterium psychrophilum were present. Our goal was to monitor the presence of these pathogens at Washington and Idaho farm sites. We achieved this for the summer and winter samples. During the initial analysis of our data, we decided that the low presence of these pathogens necessitated the development of a more sensitive assay using droplet digital PCR. We were able to develop this assay for F. columnare and are currently adapting it for F. psychrophilum. Our analysis suggests that both pathogens can be detected in water and in fish samples. Under Objective 3.b, we used a comparative genomics approach to identify toxins secreted via the type three secretion system (T3SS) of Aeromonas. A total of 105 genomes were used in this comparison, including 40 newly-sequenced Aeromonas spp genomes. A new bioinformatics approach was developed to identify secreted T3SS toxins; this approach relied on grouping the genes into orthologous groups. The 25,518 gene families were compared to the known T3SS toxins. All 633 positive gene families were compared to genomes from organisms that do not possess T3SS to remove false negatives. This screen resulted in 127 gene families that were manually curated and yielded 21 putative effectors. Each effector was cloned into an expression vector and expressed in Saccharomyces cerevisiae to assess its toxicity. Of the 21 assessed proteins, 17 were toxic to eukaryotic cells, strongly suggesting that they are T3SS toxins. Thirteen of these gene families had not been reported in Aeromonas before. This study lays the foundation for investigating the role of these toxins in virulence in fish.
Accomplishments
1. Development and commercialization of a Lactococcus vaccine for rainbow trout. Lactococcus garvieae infection is a major cause of on-farm loss of rainbow trout in Washington State. ARS researchers at Leetown, West Virginia successfully developed a vaccine against L. garvieae and validated the safety and efficacy of a commercially-manufactured version of the vaccine. The commercial vaccine is in large-scale use at affected farm sites with 6 million fish vaccinated since 2015. Field evaluation results demonstrated that vaccination induced a strong antibody response and robust protection against experimental pathogen exposure. Mortality due to L. garvieae was dramatically reduced the first year after vaccination and the disease has not been detected in vaccinated fish since program initiation. The rapid development and implementation of a Lactococcus vaccine prevented substantial rainbow trout losses due to this emerging disease.
2. Development of a new assay for measuring immune system activation in rainbow trout. Infectious disease causes appreciable losses in aquaculture, and knowledge of the immune response is incomplete. ARS researchers at Leetown, West Virginia developed a rapid and standardized assay that simultaneously measured multiple immune genes that were identified from the recently sequenced rainbow trout genome. Using this assay, fifteen genes were identified that exhibited altered expression following pathogen exposure. Analysis of a commercially-available disease-resistant rainbow trout line developed by ARS, and comparison to a reference susceptible line identified three genes that contribute to a survival difference between lines. These findings have allowed fish health workers to rapidly measure the inflammatory response and identify genes associated with disease resistance, and provides a new means for evaluating fish health on-farm.
Review Publications
Paneru, B., Al Tobasei, R., Palti, Y., Wiens, G.D., Salem, M. 2016. Differential expression of long non-coding RNAs in three genetic lines of rainbow trout (Oncorhynchus mykiss) in response to infection with Flavobacterium psychrophilum. Scientific Reports. 6:36032 doi: 10.1038/srep36032.
Kutyrev, I., Cleveland, B.M., Leeds, T.D., Wiens, G.D. 2016. Proinflammatory cytokine and cytokine receptor gene expression kinetics following challenge with Flavobacterium psychrophilum in resistant and susceptible lines of rainbow trout (Oncorhynchus mykiss). Fish and Shellfish Immunology. 58:542-553. doi: 10.1016/j.fsi.2016.09.053.
Kutyrev, I., Cleveland, B.M., Leeds, T.D., Wiens, G.D. 2017. Dataset of proinflammatory cytokine and cytokine receptor gene expression in rainbow trout (Oncorhynchus mykiss) measured using a novel GeXP multiplex, RT-PCR assay. Data in Brief. 11:192-196.
Zwollo, P., Hennessey, E., Moore, C., Marancik, D.P., Wiens, G.D., Epp, L. 2017. A BCWD-resistant line of rainbow trout exhibits higher abundance of IgT+ B cells and heavy chain tau transcripts compared to a susceptible line following challenge with Flavobacterium psychrophilum. Developmental and Comparative Immunology. 74:190-199.
Kumar, G., Hummel, K., Ahrens, M., Menanteau-Ledouble, S., Razzazi-Fazeli, E., Welch, T.J., El-Matbouli, M. 2016. Shotgun proteomic analysis of Yersinia ruckeri isolates under normal and iron-limited conditions. Veterinary Research. 47:100. doi:10.1186/s13567-016-0384-3.
Verner-Jeffreys, D., Brazier, T., Perez, R.Y., Ryder, D., Hoare, R., Welch, T.J., Ngao, T., Card, R., Mclaren, N., Ellis, R., Rowe, W., Bartle, K. 2017. Detection of the florfenicol resistance gene floR in Chryseobacterium isolates from rainbow trout. Exception to the general rule? FEMS Microbiology Ecology. 93(4):fix015. doi:10.1093/femsec/fix015.
Welch, T.J., Goodrich, T.D., La Patra, S.E. 2017. Efficacy testing of 35-year-old commercially-produced ERM bacterin reveals the remarkable stability of this product. Journal of Fish Diseases. 00:1–4. doi:10.1111/jfd.12646.
Snyder, A.K., Graf, J., Welch, T.J. 2016. The flagellar master operon flhDC is a pleiotropic regulator involved in motility and virulence of the fish pathogen Yersinia ruckeri. Journal of Applied Microbiology. 122(3):578-588. doi: 10.1111/jam.13374.
Evenhuis, J., La Patra, S.E., Graf, J. 2017. Draft genome sequence of the fish pathogen Flavobacterium columnare strain CSF-298-10. Genome Announcements. 5(15):e00173.17. doi:10.1128/genomeA.00173-17.
