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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

2015 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:
This is the first year of a five-year project that has three major objectives. Objective 1 Progress: Ninety-eight resistant- (ARS-Fp-R), 30 control- (ARS-Fp-C), and 21 susceptible-line (ARS-Fp-S) trout families were produced and evaluated at approximately 3.0 grams for bacterial cold water disease resistance (BCWD) in a standardized 21-day laboratory challenge. Mean family survival rates were 84.9% (ARS-Fp-R), 50.5% (ARS-Fp-C), and 22.0% (ARS-Fp-S). Through five generations, selection response in the ARS-Fp-R line has averaged approximately +12 percentage points (survival rate) per generation when estimated using genetic trend or comparison with randomly-mated control lines. A total of 42 families were released in 2015 to two major industry stakeholders. Approximately 320,000 embryos from the ARS-Fp-R line were distributed to Idaho stakeholders for the purposes of both small-scale and continued large-scale farm trials. One-hundred ARS-Fp-R line families were evaluated for columnaris disease resistance using a waterborne challenge and family survival averaged 84.9% (range 52.1% to 100%). Mean columnaris survival of mixed-family pools was 72.4% in our BCWD-control line (ARS-Fp-C) and 50.4% in our BCWD-susceptible line (ARS-Fp-S). Previously, we mapped quantitative trait loci (QTL) for BCWD resistance and spleen size to rainbow trout chromosome Omy19 utilizing microsatellite markers. In collaboration with Research Project 8082-31000-012, we utilized a new technology, RAD-seq (restriction-site-associated DNA sequencing) to sequence DNA from 298 offspring from two half-sib families used in our previous study. From the sequencing, a total of 7,849 informative SNPs were identified in the two families. Using genome wide association analyses, 18 SNPs were associated with BCWD resistance and 20 SNPs associated with spleen size. Mapping revealed three significant QTL for BCWD resistance. In addition to the previously validated QTL on chromosome Omy19, two significant BCWD QTL were identified on chromosomes Omy8 and Omy25. A significant QTL for spleen size was identified on chromosome Omy2. These SNP markers will facilitate further fine mapping to identify positional candidate genes for BCWD resistance. Over 200 individual Flavobacterium columnare isolates were collected from aquaculture farms located along the Snake River in southern Idaho. All isolates collected to date belong to genomovar I. Eleven isolates were further characterized by virulence phenotyping, amplified fragment length polymorphism (AFLP) analysis and draft genome sequencing. Virulence ranged from an LD50 of 1x106 CFU mL-1 to 8 x106 CFU mL-1. AFLP analysis identified that the most virulent strain was also the most evolutionarily divergent. Draft genome sequencing has been completed, and nucleotide variation between strains is being analyzed. These results indicated F. columnare sequence heterogeneity in southern Idaho and also variation in strains within an individual farm. Objective 2 Progress: Methods have been developed for quantifying the expression of Yersinia ruckeri flagellar secretion products using reverse transcription polymerase chain reaction assays. These assays have been used to examine flagellin gene expression during the infection process. A flhDC transcriptional reporter system has been developed and will be used to screen for Y. ruckeri genes that regulate flhDC expression in an effort to identify regulators of the flagellar motility system. We have also created a fliC reporter system capable of assessing flagellin expression visually using flow cytometry or microscopy. Objective 3 Progress: We reported the complete genome sequence of Yersinia ruckeri strain CSF007-82 and two associated plasmids. High quality RNA has been isolated from Y. ruckeri cells and transcriptome analyses are underway. Disease outbreak investigation has been completed at a major stakeholder and 12 Aeromonad isolates have been draft sequenced. This information is being utilized to select and analyze autogenous vaccine performance. We published one manuscript that established a bioinformatics approach to identify Aeromonas species in mBio. We are currently performing bioinformatics comparisons, which will lead to the publication of six of these genomes. We are in the process of establishing the cell culture assay for virulence.

4. Accomplishments
1. Determination of the complete genome sequence of biotype 1, Yersinia ruckeri strain CSF007-82. Yersinia ruckeri is a reemerging pathogen of farmed rainbow trout and increased understanding of virulence is needed to develop better control methods. ARS scientists at the Leetown, West Virginia, worksite and collaborators at the University of Connecticut completely sequenced the 3,799,036 bp chromosome encoding 3,517 predicted coding sequences as well as two plasmids named pYR2 and pYR3. The availability of this complete and finished genome sequence has facilitated the analysis of virulence factors associated with Y. ruckeri-caused disease and will enable studies of the evolution of this important pathogen.

Review Publications
Colston, S., Fullmer, M., Beka, L., Lamy, B., Gogarten, J.P., Graf, J. 2014. Bioinformatic genome comparisons for taxonomic and phylogenic assignments using Aeromonas as a test case. mBio. 5(6):e02136-14. DOI:10.1128/mBio.02136-14.