2007 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 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 report serves to document research conducted under a Non-Funded Cooperative Agreement between ARS and Clear Springs Foods Inc.(1930-32000-001-03N). Bacterial cold water disease, caused by Flavobacterium psychrophilum, is a serious problem for rainbow trout aquaculture. Conventional approaches to vaccine development have been ineffective and antimicrobial resistance is of concern. This year we reported the completed genome of Flavobacterium psychrophilum. A PCR assay was developed to distinguish between genes containing repeated sequences and this assay was used to show that one region is highly variable between strains. One of the genes contained within this variable region, designated F. psychrophilum repeat protein N (frpN), was cloned and a recombinant protein was purified. This protein did not induce a protective immune response in rainbow trout. The complete genome sequence is the first step toward a better understanding of metabolic and pathogenic processes, and the identification of vaccine candidates. The completion of the genome sequence was the motivation for a workshop, Flavobacterium 2007, held in Shepherdstown WV on May 2-4th that was attended by over 70 scientists from eight countries. This work also addresses research outlined in the National Program 106 Action Plan. Integrated Aquatic Animal Health. Goal 1. Microbial Genomics.
Title: Characterization of a multiple drug resistance (MDR) plasmid from Yersinia ruckeri.
Problem statement: The emergence and dissemination of mobile antimicrobial resistance among pathogenic and commensal bacteria associated with animal production systems is a significant concern. An improved understanding of the evolution and ecology of mobile antimicrobial resistance elements is essential for both predicting the utility of antimicrobial drugs and understanding the risks associated with their use.
Accomplishment: A multiple drug resistance (MDR) plasmid was identified in Y. ruckeri and completely sequenced. This plasmid was shown to be highly related to MDR plasmids present in clinical isolates of Yersinia pestis and Salmonella enterica. The high degree of sequence identity and gene synteny among these plasmids suggests recent dissemination from a common ancestor. Subsequent screening has shown that this plasmid type is currently broadly disseminated among several human and animal pathogens isolated from retail meat samples. Impact: These plasmids represent a reservoir of mobile resistance determinants and have the potential to disseminate to Y. pestis and other human and zoonotic bacterial pathogens of public health importance. We have also developed rapid detection and typing methods that could be used to monitor this antimicrobial resistance plasmid in animal production systems, foods, and may be useful for detecting these plasmids in biothreat organisms. Identification would be the first step toward reducing the prevalence of these MDR conferring plasmids. National Program Component and Problem Statement. National Program 106 Action Plan, Integrated Aquatic Animal Health Management: Epidemiology. Goal 1. Develop methods to assess risk factors associated with the economically important pathogens of aquatic animals.
Title: Identification of new rainbow trout immune genes.
Problem statement: Infectious disease is a significant factor hindering aquaculture and a better understanding the fish immune system is required to improve health and disease resistance. Accomplishment: In this study, we identified forty-four new tumor necrosis family gene sequences from rainbow trout and other fish species. Comparison of the protein sequences and chromosomal locations allowed us to identify which genes were related to those found in mammals and which were unique to fish. We developed methods for measuring gene expression in rainbow trout tissues. Impact: These studies have led to a better understanding of how the fish immune system functions and how it differs from that of mammals. The newly developed gene detection methods will advance studies on gene expression and functional analyses in fish. National Program Component and Problem Statement: National Program 106 Action Plan, Integrated Aquatic Animal Health Management: Immunology and Disease Resistance. Goal 1. Develop methods to characterize cells and regulatory substances (cytokines) important in natural resistance and acquired immunity in aquatic animals.
Bebak, J.A., Welch, T.J, Starliper, C.E., Baya, A.M., Garner, M.M. 2007 Improved husbandry to control an outbreak of rainbow trout fry syndrome caused by infection with Flavobacterium psychrophilum. Joural of the American Veterinary medical Asociation. 231: 114-116.
5.Significant Activities that Support Special Target Populations
|Number of new CRADAs and MTAs||2|
|Number of active CRADAs and MTAs||4|
|Number of invention disclosures submitted||1|
|Number of web sites managed||1|
|Number of non-peer reviewed presentations and proceedings||8|
|Number of newspaper articles and other presentations for non-science audiences||26|
Welch, T.J., Fricke, W.F., Rosso, M.L., Rasko, D.A., Mammel, M., Eppinger, M., Rosovitz, M.J., White, D.G., Mcdermott, P.F., Wagner, D., Rahalison, L., Leclerc, J.E., Hinshaw, J.M., Lindler, L.E., Cebula, T.A., Carniel, E., Ravel, J. 2007. Multiple Antimicrobial Resistance in Plague: An Emerging Public Health Risk. PLoS One. Vol.1 (3)1-6.
Glenney, G.W., Wiens, G.D. 2007. Early Diversification of the Tumor Necrosis Superfamily in Teleosts: Genomic Characterization and Expression Analysis. Journal of Immunology, 178:7955-7973.
Stork, M., Di Lorenzo, M., Welch, T.J., Crosa, J. 2007. Transcription termination Within the iron transport-biosynthesis operon of Vibrio anguillarum requires an antisense RNA. Journal of Bacteriology 189:(9), 3479-88.
Sudheesh, P.S., Lafrentz, B.R., Call, D.R., Siems, W.F., Lapatra, S.E., Wiens, G.D., Cain, K.D. 2007. Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum. Diseases of Aquatic Organisms. 74(1):37-47.