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United States Department of Agriculture

Agricultural Research Service

Research Project: HOST, PATHOGEN AND ENVIRONMENTAL INTERACTIONS IN COOL AND COLD WATER AQUACULTURE
2008 Annual Report


1a.Objectives (from AD-416)
1. Characterize virulence determinants produced by the rainbow trout pathogens Yersinia ruckeri and Flavobacterium psychrophilum. 2. Identify rainbow trout genes and factors that are critical to expression of innate and acquired immunity against Y. ruckeri and F. psychrophilum. 2a. Molecular identification and sequencing of antibiotic resistance and virulence genes in bacterial pathogens of aquaculture species. 3. Identify water quality factors that influence host susceptibility and pathogen refuge within a recirculating system, and evaluate the use of bacteriophage as a targeted antibacterial strategy in recirculating and serial reuse aquaculture systems.


1b.Approach (from AD-416)
Virulence determinants and antibiotic resistance plasmids of Yersinia ruckeri and Flavobacterium psychrophilum will be identified using molecular genetic approaches. Y. ruckeri virulence factors will be established by screening transposon-induced mutants for attenuation of virulence. For F. psychrophilum, we will completely sequence the genome and target potential virulence genes for mutation. These studies will generate candidates for vaccines and rapid diagnostic assays. Host factors involved in innate and acquired immunity will be identified by bioinformatics and gene expression analyses. The expression of trout immune genes will be measured in vaccinated and challenged fish by RT-PCR and immune gene microarrays. These studies will identify targets for diagnostic assays measuring protective immunity. The impact of organic constituents on host disease susceptibility and microbial growth will be assessed in recirculating aquaculture systems. Pathogen refuges within culture systems will be identified to aid targeted disinfection strategies. These multidisciplinary efforts will define the important parameters governing infectious disease in order to develop effective vaccines, novel biotherapeutics and strategies for maintaining healthier fish rearing conditions for the aquaculture industry.


3.Progress Report
Bacterial coldwater disease is the major bacterial pathogen affecting trout aquaculture. As part of a multifaceted approach to disease control, we have previously shown the resistance is a heritable trait and that resistance can be improved by breeding. This year, we evaluated families of relative resistant and susceptible fish crosses (Brood-year 2007) for resistance against diverse strains of F. psychrophilum. Resistant fish performed better against all tested strains suggesting broad-ranged coldwater disease resistance exists in our broodstock. We also evaluated vaccine prototypes in resistant and susceptible crosses of fish. Research combining vaccination with selective breeding is underway with the goal of enhancing both the innate and acquired immune response. (NP 106-Aquaculture; Animal Health; Immunology and Disease Resistance.)

Enteric red mouth disease in salmonid aquaculture has been successfully controlled by immersion vaccination for over two decades. However, an emergent biotype of Yersinia ruckeri (biotype II) has caused recent outbreaks in vaccinated fish in both the United States and Europe. Complementation, sequence and mutation analyses were used to identify the primary genetic differences that define this new Y. ruckeri biotype. Research elucidating the mechanism(s) causing this apparent vaccine failure is in progress. (NP 106-Aquaculture; Integrated Aquatic Animal Health Management: Mechanism of Disease and Microbial Genomics.)

The fish immune response to Yersinia ruckeri is poorly understood. Rainbow trout were challenged with Y. ruckeri and bacterial numbers and fish gene expression were measured. Temporal and dose-dependent changes in gene expression were identified over a 7 day challenge period. Analysis of gene expression patterns and correlation with infection severity is ongoing. (NP 106-Aquaculture; Animal Health; Immunology and Disease Resistance.)

Multiple antimicrobial resistance plasmids cause critical agricultural and human health problems. This year, two resistance plasmids of aquaculture origin have were identified and partially characterized by DNA sequence analysis. Both plasmids were shown to be conjugative and encode resistance to multiple antimicrobial agents including all three antimicrobial drugs approved for use in aquaculture. (NP 106-Aquaculture; Integrated Aquatic Animal Health Management: Mechanism of Disease and Microbial Genomics.)

Bacteriophage have potential as safe and effective antimicrobials that offer the aquaculture industry an alternative to traditional antibiotics. Using a bacteriophage specific to the fish pathogen Y. ruckeri we have identified a novel bacteriophage-produced enzyme activity that neutralizes the virulence of this pathogen by degrading bacterial components that are essential for survival during infection. (NP 106-Aquaculture; Development of safe and effective vaccines and medicines for prevention and control of economically important pathogens of aquatic animals.)


4.Accomplishments
1. Selection marker of rainbow trout disease resistance identified. Infectious disease is a substantial source of loss in US rainbow trout aquaculture and improved methods are needed to diminish this problem. At the NCCCWA, we have selectively bred rainbow trout for increased resistance to the bacterial cold-water disease agent, Flavobacterium psychrophilum, demonstrating that most fish remained resistant throughout their life cycle. We also found that resistant fish crosses had, on average, a larger spleen size than susceptible fish crosses. Selecting fish crosses solely based on spleen size was found to predict resistance to the bacterial cold-water disease agent indicating a close link between these two traits. Since spleen size is easy to measure, we suggest that it may be a useful selection parameter for evaluation in other fish populations. National Program 106 Action Plan, Integrated Aquatic Animal Health Management: Immunology and Disease Resistance. Goal 4.

2. Complete genome sequence of the fish pathogen Renibacterium salmoninuarm determined. Renibacterium salmoninarum (Rs) is the causative agent of bacterial kidney disease and a significant threat to the healthy and sustainable production of salmon and trout. Limited information on the pathogen is available. We determined the complete genome sequence of Rs strain ATCC 33209 and published the data as GenBank accession NC_010168. The genome is composed of a single circular chromosome comprised of over 3.1 million base pairs and 3,507 putative genes. The genome sequence has allowed the identification of therapeutic agents and vaccine candidates that may have future utility in preventing or treating infections in cultured salmonids. This accomplishment resulted from collaboration between investigators at the NCCCWA, NOAA, Oregon State University, University of Washington, and Integrated Genomics. National Program 106 Action Plan, Integrated Aquatic Animal Health Management: Microbial Genomics, Goal 1.


5.Significant Activities that Support Special Target Populations
NONE


6.Technology Transfer

Number of the New MTAs (providing only)1

Review Publications
Hadidi, S., Glenney, G., Welch, T.J., Silverstein, J., Wiens, G.D. 2008. Spleen Size Predicts Resistance of Rainbow Trout to Flavobacterium psychrophilum Challenge. Journal of Immunology. 180:4156-4165.

Last Modified: 10/25/2014
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