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ARS Home » Northeast Area » Leetown, West Virginia » Cool and Cold Water Aquaculture Research » Research » Research Project #428108

Research Project: Integrated Research to Improve On-Farm Animal Health in Salmonid Aquaculture

Location: Cool and Cold Water Aquaculture Research

2016 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
Objective 1: The odd-year line of National Center for Cool and Cold Water Aquaculture (NCCCWA) rainbow trout (ARS-Fp-R) was evaluated for variation in innate resistance to F. columnare immersion challenge in 2013 and 2015. Results from both years indicated measurable differences in F. columnare resistance between families that was determined to be both heritable (0.17 ±0.09) and favorably correlated (0.35 ±0.25) to bacterial cold water disease (BCWD) resistance. Genetic correlations were small and antagonistic (-0.15 ± 0.08 to -0.19 ± 0.24) between the 2 resistance traits and 9- and 12-month body weight. These results established the feasibility of producing a line of trout that has improved resistance against both BCWD and columnaris disease. The first generation of selection has been applied to families from the 2015 year-class. In 2015 a production scale farm-trial was initiated, and 300,000 eyed-eggs from the ARS-Fp-R line shipped from the NCCCWA to a commercial farm-site in Idaho. The performance of the fish were followed from hatching through harvest. BCWD resistance was confirmed by laboratory exposure at 80 days post-hatch in a cohort of fish, and no outbreak of BCWD was observed at the farm site in the production lot. BCWD was present at the facility and diagnosed in five of eight contemporary lots that exhibited elevated mortality. During hatch-house rearing, ARS-Fp-R line was vaccinated against infectious hematopoietic necrosis virus (IHNV) and subsequently vaccination efficacy confirmed by laboratory challenge. In contrast to the 2013 production-scale trial, no IHNV outbreak occurred during the 2015 trial. However, limited gill disease losses occurred between 264 and 287 days post-hatch resulting in 3% cumulative mortality. Growth and feed conversion, as assessed at trial completion, were within acceptable parameters. Throughout the grow-out period, water quality was continuously measured and serum biochemistry samples collected at two time points. Analyses of water quality data and plasma biochemistry samples are on-going. We continue to add genotyped F. psychrophilum and F. columnare isolates to a database of strains associated with rainbow trout farm-site disease outbreaks. A total of eleven F. columnare genomovar I isolates and 13 isolates from other genomovars were draft genome sequenced, by MiSeq, and assembled using both VELVET and CLCBio software. A high-quality genome sequence of F. columnare CSF298-10 is underway. Objective 2: To better understand flagellar and virulence regulation in Y. ruckeri an isogeneic mutant lacking the flhD gene was constructed and used to demonstrate its role in flagellar biosynthesis. In addition, efforts to identify genes which influence flhD expression resulted in the identification of an environmental response-regulator which controls in vivo expression of the flagellar biosynthesis system and thereby regulates virulence in Y. ruckeri. RNA-seq analysis suggests a global regulatory system involved in host sensing and virulence regulation. Objective 3: Disease outbreak investigations at a trout farm in Washington State have revealed Lactococcus garvieae as a major cause of disease loss. We have developed and validated an autogenous vaccine against this pathogen and are in the process of validating the efficacy of this vaccine under field conditions. Microbiome analysis at producer sites in Washington and Idaho are in progress. These studies will allow us to monitor the microbiome of aquaculture sites over time tracking changes in microbial community composition in relation to outbreaks of disease. We have also sequenced the genomes of 12 additional Aeromonas veronii strains obtained from different sources and evaluated their virulence in zebrafish and wax worms and in symbiotic competence in the medicinal leech. Interestingly, strains that were more virulent in zebrafish were not necessarily highly virulent in wax worms, which is consistent with the involvement of different virulence factors in different associations. We have also established a cell culture model that allows us to detect damage to fish cells after exposure to bacteria. We used two different cell lines, RTG from Oncorhynchus mykiss and EPC from Papulosum Cyprini, with different growth temperature optima, 18ºC and 25ºC respectively. The development of this virulence assay will allow us to test strains quickly for virulence characteristics and pursue identification of the underlying molecular mechanisms.

4. Accomplishments
1. Genetic diversity of Flavobacterium psychrophilum isolates from the United States determined by multilocus sequence typing. Bacterial cold water disease (BCWD) is a frequent cause of freshwater farmed trout loss and genetic diversity of the pathogen is poorly understood. Scientists at the National Center for Cool and Cold Water Aquaculture (NCCCWA) in Leetown, West Virginia in collaboration with scientists at the College of Veterinary Medicine, Michigan State University, and French National Institute for Agricultural Research (INRA), France typed 96 isolates of F. pyschrophilum recovered from rainbow trout, coho salmon, and Chinook salmon that originated from nine U.S. states. Multilocus sequence typing identified 34 types that clustered into 5 groups. Sequence type 10 was commonly associated with bacterial cold water disease outbreaks at rainbow trout farms. This information improves understanding of genetic diversity and strains associated with disease outbreak and will help direct targeted vaccines and improve selection of disease resistant rainbow trout.

2. Yersinia ruckeri lipopolysaccharide is necessary and sufficient for eliciting a protective immune response in rainbow trout. A highly effective vaccine was developed in the 1970's to prevent infection caused by the bacterial pathogen Yersinia ruckeri. The unusual success of this vaccine has led to the use of Y. ruckeri vaccination as a model system for better understanding immersion vaccination. While much has been learned regarding host response to Y. ruckeri vaccination the bacterial components necessary for eliciting this protective response remain unclear. ARS scientists at the National Center for Cool and Cold Water Aquaculture in Leetown, West Virginia have demonstrated that highly purified Y. ruckeri lipopolysaccharide (LPS) alone is a highly potent immunogen and is sufficient for eliciting a strong protective response. We also created a defined Y. ruckeri mutant lacking LPS and used this mutant to demonstrate that LPS is an essential component of the whole cell vaccine. Together these results suggest that LPS is the only cellular component contributing to the protective response elicited by the Y. ruckeri bacterin vaccine. We propose that the exceptionally high potency of Y. ruckeri LPS accounts for the unusual success of this vaccine when delivered by immersion. This work contributes to a better understanding of Y. ruckeri vaccination by identifying the bacterial factors necessary for eliciting a protective response.

3. Genome sequencing of Lactococcus garvieae strain PAQ102015-99, an outbreak strain identified from cultured rainbow trout in the northwestern United States. L. garvieae, the causative agent of lactococcosis, is a commercially important pathogen of farmed rainbow trout. ARS scientists at the National Center for Cool and Cold Water Aquaculture in Leetown, West Virginia have recently identified an outbreak of lactococcosis at a commercial trout aquaculture facility in Washington State and have determined the draft genome sequence of a representative strain. This information will be critical for the development of strain-specific diagnostics and for the identification of virulence factors and surface characteristics.

5. Significant Activities that Support Special Target Populations:
“Production-scale farm evaluation of the ARS-Fp-R line of bacterial cold water disease resistant rainbow trout”. World Aquaculture Society, Aquaculture America, Trout Farmers Session, Las Vegas, NV, Feb. 22-26, 2016. Target Population: General Public, Small Producers and Scientists. G.D. Wiens, C. Birkett, T. Leeds, S. LaPatra.

Review Publications
Van Vliet, D., Wiens, G.D., Loch, T.P., Nicolas, P., Faisal, M. 2016. Genetic diversity of Flavobacterium psychrophilum isolates from three Oncorhynchus spp. in the United States, as revealed by multilocus sequence typing. Applied and Environmental Microbiology. 82:3246-3255.

Liu, S., Vallejo, R.L., Palti, Y., Gao, G., Marancik, D.P., Hernandez, A.G., Wiens, G.D. 2015. Identification of single nucleotide polymorphism markers associated with bacterial cold water disease resistance and spleen size in rainbow trout. Frontiers in Genetics. 6:298. doi:10.3389/fgene.2015.00298.

Snyder, A.K., Hinshaw, J.M., Welch, T.J. 2015. Diagnostic tools for rapid detection and quantification of Weissella ceti NC36 infections in rainbow trout. Letters in Applied Microbiology. DOI: 10.1111/lam.12365.

Good, C., Davidson, J., Wiens, G.D., Welch, T.J., Summerfelt, S. 2014. Flavobacterium branchiophilum and F. succinicans associated with bacterial gill disease in rainbow trout Oncorhynchus mykiss (Walbaum) in water recirculation aquaculture systems. Journal of Fish Diseases. DOI: 10.1111/jfd.12249.

Good, C., Marancik, D.P., Welch, T.J., May, T., Davidson, J., Summerfelt, S. 2015. Systemic granuloma observed in Atlantic salmon Salmo salar raised to market size in a freshwater recirculation aquaculture system. Aquaculture Research. DOI: 10.1111/are.12790:1-5.

Evenhuis, J., Mohammad, H., Lapatra, S.E., Welch, T.J., Arias, C. 2016. Virulence and molecular variation of Flavobacterium columnare affecting rainbow trout in ID, USA. Aquaculture. 464:106-110. doi: 10.1016/j.aquaculture.2016.06.017.

Mosser, T., Talagrand, E., Colston, S.M., Graf, J., Figueras, M., Jumas-Bilak, E., Lamy, B. 2015. Exposure to pairs of Aeromonas strains enhances virulence in the Caenorhabditis elegans infection model. Frontiers in Microbiology. 6:1218. doi: 10.3389/fmicb.2015.01218.

Nelson, M.C., Lapatra, S.E., Welch, T.J., Graf, J. 2015. Complete genome sequence of Yersinia ruckeri str. CSF007-82, etiologic agent of enteric redmouth disease in salmonid fish. Genome Announcements. 3(1):e01491-14. DOI:10.1128/genomeA.01491-14.

Welch, T.J., Lapatra, S. 2016. Yersinia ruckeri lipopolysaccharide is necessary and sufficient for eliciting a protective immune response in rainbow trout (Oncorhynchus mykiss, Walbaum). Fish and Shellfish Immunology. 49:420-426. doi:10.1016/j.fsi.2015.12.037