2010 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.
This is the final report for 1930-32000-002-00D which terminated December 8, 2009. Project was replaced by 1930-32000-005-00D. Accomplishments are listed in the replacement project.
An engineered strain of biotype 2 Y. ruckeri was developed which constitutively expresses the green fluorescent protein (GFP). GFP expression was exceptionally stable and the strain retained its ability to colonize and cause disease in immersion-challenged rainbow trout. This strain has been used for detecting and imaging the interactions between this pathogen and its natural salmonid host. A standardized disease challenge model has been established with this strain.
As part of a multifaceted approach to disease control, we determined variation in survival between families, heritability of post-challenge survival, the stability of resistance during growth, and the response to selection. Over 10,000 fish from 200 families were evaluated by 21 day challenge. The heritability of post-challenge survival, an indicator of disease resistance, was estimated to be 0.35 ± 0.09 and there was no genetic or phenotypic correlation with growth rate, or the 9 and 12 month body weights. Fish maintained their relative “resistant” or “susceptible” phenotype as average body weight increased over 300-fold. These results demonstrate that the mechanism(s) responsible for the higher survival are not transitory during fish development and suggest that our findings are relevant to a wide range of rainbow trout sizes. These results demonstrated that selective breeding is an effective approach to improving resistance to bacterial cold water disease challenge in rainbow trout.
A major need in selective breeding is the development of easy-to-measure surrogates of disease resistance. We demonstrated 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 link between these two traits. Since spleen size is easy to measure, it may be a useful selection parameter for evaluation in other fish populations.
A number of novel rainbow trout immune genes were discovered and described that belong to the chemokine, tumor necrosis factor, and toll-like receptor families of immune genes. We reported the discovery of two new rainbow trout chemokine genes: CXCd1 and CXCd2. A comprehensive analysis of the TNF superfamily in teleost fish was completed. Forty-two novel genes were identified from database searches and sequencing efforts.