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)
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.