Source: LOUISIANA STATE UNIVERSITY submitted to NRP
INTRACELLULAR PATHOGENESIS OF EDWARDSIELLA ICTALURI
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0217395
Grant No.
2009-35204-05312
Cumulative Award Amt.
(N/A)
Proposal No.
2008-00884
Multistate No.
(N/A)
Project Start Date
Mar 15, 2009
Project End Date
Mar 14, 2012
Grant Year
2009
Program Code
[44.0A]- Animal Protection & Biosecurity (A): Animal Disease
Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
Pathobiological Sciences
Non Technical Summary
Commercial catfish production accounts for 85-90% of the total fin fish aquaculture production in the United States, with almost 300,000 tonnes produced annually. Significant losses due to the bacterial pathogen Edwardsiella ictaluri were reported on over 60% of all farms in operation. The proposed project addresses fundamental research on the pathogenesis of E. ictaluri. Based on significant preliminary data that was carefully analyzed and integrated with the known scientific literature, a model for E. ictaluri pathogenesis was developed. Confirmation of the model, or even portions of the model, will provide information that will lead to conceptual breakthroughs in management procedures to control ESC. By testing a series of scientific hypotheses, this model will be carefully evaluated. Elucidation of the intracellular location and function of the E. ictaluri virulence factors will lead to knowledge of their role in pathogenesis and their possible interaction with the host immune system. Ultimately this knowledge could be used to influence the catfish immune response to E. ictaluri infections and provide a basis for construction of vaccines that would lead to the preventions of E. ictaluri infections in channel catfish.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113710110050%
3114010110050%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4010 - Bacteria; 3710 - Catfish;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Work to identify virulence factors of E. ictaluri using signature-tagged-mutagenesis has identified several genes that encode proteins that have a strong likelihood of involvement in intracellular replication, including arginine decarboxylase (AdiA), a urease complex, and an E. ictaluri type III secretion system (T3SS). Phenotypic characterization of strains carrying mutations in those genes confirmed that each are required for virulence in channel catfish and for successful survival and replication within channel catfish head-kidney derived macrophages (HKDM). Analysis of the putative biological activities of AdiA, urease, the T3SS, along with other genes associated with their activity, enabled the development of a unique preliminary model to explain the mechanisms associated with intracellular survival and replication of E. ictaluri. The model is strongly supported by the literature and by preliminary data. The primary objective of this proposal is to confirm the validity of the E. ictaluri pathogenesis model. Consequently, we propose two specific aims to test the model and define the role of the identified genes in the intracellular pathogenesis of E. ictaluri. Specific Aim 1. Define the role of arginine cycling in E. ictaluri pathogenesis. Specific Aim 2. Define the role of the type III secretion system in E. ictaluri pathogenesis.
Project Methods
As described above, a model for E. ictaluri survival and replication in catfish macrophages was developed. The general approach to confirming this model is to answer questions concerning transcription and translation of the genes involved, and concerning the phenotype associated with transcription and translation of those genes, using defined mutants to evaluate changes that are associated with gene loss. All fish used in these experiments will be from channel catfish egg masses obtained from a commercial producer with no history of ESC outbreaks. Eggs will be disinfected with 100 ppm free iodine and hatched in closed recirculating systems in the specific pathogen free laboratory at the LSU School of Veterinary Medicine. Experimental designs will be completely randomized with a factorial arrangement of treatments. Time course experiments and differences between wild-type and mutant strains will be analyzed using the General Linear Model from Statistical Analysis Systems (SAS) version 9.1 (SAS Institute, Incorporated, Cary, North Carolina). When the overall model indicates significance at p ≤ 0.05, the Least Significant Difference test will be used for pair wise comparison of main effects, and a least square means procedure will be used for pair-wise comparison of interaction effects. When cfu numbers or percentage are analyzed, data will be subjected to a log transformation and adjusted to a positive integer scale. Gene expression will be assessed by reverse transcriptase PCR (RT-PCR). Expression of specific genes will be assessed by PCR using gene specific primers and Phusion High Fidelity DNA Polymerase (NEB, La Jolla, CA). Where quantification of specific gene expression is important, transcripts will be detected using quantitative real time PCR (Q-PCR). A reaction containing cDNA, master mix, primers and probe will be made for specific genes and Q-PCR will be performed and analyzed using the ABI 7900HT Real Time PCR System and SDS Enterprise software (Applied Biosystems, Foster City, CA). E. ictaluri 16S ribosomal RNA will be amplified to normalize specific gene expression and mean threshold cycle (Ct) values will be calculated. Western blotting will be used to detect translation of specific proteins. In order to provide templates for mutant construction, pBKCMV plasmids carrying the specific genes of interest are isolated from our previously described lambda-library and sequenced for confirmation. Individual gene mutations will be constructed by overlapping-primer extension PCR (OPE-PCR). Completion of this project will provide a basic understanding of how E. ictaluri survives and replicates in catfish macrophages that will be disseminated to the scientific community through peer reviewed publications. Knowledge of the mechanisms and the specific virulence factors involved will enable development of new procedures for the prevention and control of E. ictaluri infections in channel catfish that will be disseminated to the catfish farming industry through Cooperative Extension.

Progress 03/15/09 to 03/14/12

Outputs
OUTPUTS: Seven presentations were given on various aspects of the project at annual meetings of the Fish Health Section of the American Fisheries Society, including four at the International Symposium on Aquatic Animal Health in Tampa, FL in 2010. Summaries of the model for E. ictaluri pathogenesis as developed to this point were presented at the Catfish Farmers of America Research Symposium in Savannah, GA on 2/17/12 and at the World Congress of Vaccine 2012 in Beijing, China on 3/28/12. PARTICIPANTS: Dr. Ron Thune Principal investigator. Research supervision, data analysis and publication production. Dr. Matt Rogge. Post-Doctoral fellow conducted work on the role of the E. ictaluri Type Three Secretion System in pathogenesis. Dr. Wes Baumgartner. Graduate research associate conducted work on the role of the E. ictaluri urease enzyme in pathogenesis. Judith Beekman. Research associate conductded work on the role of the ammonia and urea transporters in E. ictaluri pathogenesis. Dr. Lydia Dubytska, a post-doctoral fellow completed work on the acidification of the Edwardsiella containing vacuole. TARGET AUDIENCES: Presented the current model for E. ictaluri pathogenesis to the Catfish Farmers of America and catfish research scientists at the Catfish Farmers of America Research Symposium in Savannah, GA on 2/18/12. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The findings, results and techniques developed from this project were used to define a model for E. ictaluri pathogenesis that explains how the bacterium is able to survive and replicate in the hostile environment of a macrophage. Data showing coordinated regulation of two separate secretions systems has led to an expanded interpretation of the model that may lead to unique opportunities to develop new treatment and prevention modalities.

Publications

  • Baumgartner, W. 2011. Arginine metabolism in the Edwardsiella ictaluri-channel catfish macrophage dynamic. A dissertation completed at Louisiana State University. Baumgartner, W., L. Dubytska, M. Rogge, and R. L. Thune. 2012. Manipulation of the Edwardseilla ictaluri containing vacuole in channel catfish head kidney derived macrophages. In preparartion.


Progress 03/15/10 to 03/14/11

Outputs
OUTPUTS: Presented 4 talks at the 6th International Symposium on Aquatic Animal Health, Tampa, FL. September 5-9, 2010. Submitted a manuscript to Applied and Environmental Microbiology. PARTICIPANTS: Dr. Ron Thune Principal investigator. Research supervision, data analysis and publication production. Dr. Matt Rogge. Post-Doctoral fellow conducting work on the role of the E. ictaluri Type Three Secretion System in pathogenesis. Dr. Wes Baumgartner. Graduate research associate conducting work on the role of the E. ictaluri urease enzyme in pathogenesis. Judith Beekman. Research associate conducting work on the role of the ammonia and urea transporters in E. ictaluri pathogenesis. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Discovery of co-regulation of two separate secretion systems during E. ictaluri pathogenesis has expanded the work to include the second system.

Impacts
Early results have generally validated the model that was proposed for Edwardsiella ictaluri pathogenesis. Data showing coordinated regulation of two separate secretions systems has led to an expanded interpretation of the model that may lead to unique opportunities to develop new treatment and prevention modalities.

Publications

  • Beekman, J and RL Thune. 2010. Evaluation of the Role of Ammonia and Urea Transporters on the Pathogenesis of Edwardsiella ictaluri. Proceedings of the 6th International Symposium on Aquatic Animal Health, Tampa, FL. September 5-9, 2010. p. 81.
  • Rogge, M. L. and Thune, R. L. 2010. The Edwardsiella ictaluri Type III secretion system is regulated by pH and phosphate concentration through EsrA, EsrB, and EsrC. Submitted to Applied and Environmental Microbiology.
  • Graff, AA, ML Rogge and RL Thune. 2010. Evaluation of the Edwardsiella ictaluri Type IV Secretion System Role in Virulence. Proceedings of the 6th International Symposium on Aquatic Animal Health, Tampa, FL. September 5-9, 2010. p. 80.
  • Rogge, ML and RL Thune. 2010. Initial Evaluation of an Edwardsiella ictaluri Type III Secretion System Mutant as a Vaccine Candidate Against Enteric Septicemia of Catfish. Proceedings of the 6th International Symposium on Aquatic Animal Health, Tampa, FL. September 5-9, 2010. p. 85.
  • Rogge, ML and RL Thune. 2010. The Edwardsiella ictaluri Type III and Type VI Secretion Systems Are Co-Regulated and Are Both Required for Virulence in the Channel Catfish. Proceedings of the 6th International Symposium on Aquatic Animal Health, Tampa, FL. September 5-9, 2010. p. 81.


Progress 03/15/09 to 03/14/10

Outputs
OUTPUTS: Developed procedures to use SacB based positive selection to produce isogenic mutants of Edwardsiella ictaluri using the suicide plasmid pRE107. Have produced over 20 mutants to be used for the pathogenesis studies. Also developed procedures for epitope tagging of E. ictaluri mutants in order to conduct translational analysis of E. ictaluri proteins. Have tagged about 20 proteins and have preliminarily detected five of them in western blots using antibody to the tag. PARTICIPANTS: Dr. Ron Thune Principal investigator. Research supervision, data analysis and publication production. Dr. Matt Rogge. Post-Doctoral fellow conducting work on the role of the E. ictaluri Type Three Secretion System in pathogenesis. Dr. Wes Baumgartner. Graduate research associate conducting work on the role of the E. ictaluri urease enzyme in pathogenesis. Judith Beekman. Research associate conducting work on the role of the ammonia and urea transporters in E. ictaluri pathogenesis. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Production of the mutants and the tagged proteins will enable us to evaluate the E. ictaluri pathogenesis model in channel catfish macrophages.

Publications

  • No publications reported this period