Date of Award

1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

Ronald Thune

Abstract

Edwardsiella ictaluri, causative agent of enteric septicemia of catfish (ESC), is the primary bacterial pathogen of commercially produced channel catfish (Ictalurus punctatus). The purpose of this study was to make progress towards development of a successful ESC vaccine by generating and characterizing a pool of E. ictaluri antigens and determining if they were protective against ESC in catfish. Antigenic protein expression was evaluated in E. ictaluri cells under different conditions of growth using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and western blotting with pooled convalescent catfish serum (CCS). Results showed protein bands separated by one-dimensional PAGE may consist of one or more antigenic and non-antigenic proteins, and strain and culture temperatures do not effect expression of antigenic proteins, but culture media does. A 57 KD antigenic protein was only expressed by E. ictaluri grown in minimal media, and antigenic proteins of 58 and 71 KD were expressed at different levels in minimal or rich media. An E. ictaluri genomic library in an Escherichia coli expression vector was screened using goat anti-E. ictaluri serum (GAI) resulting in isolation of 32 clones expressing antigenic E. ictaluri proteins. Encoded genes and expressed antigenic proteins of nine clones were analyzed by partial DNA sequencing, 2D-PAGE and western blotting with GAI and CCS. The DNA inserts of three clones were double-strand sequenced. Four putative open reading frames were identified in the insert of clone 4d6 corresponding to antigenic proteins of 63, 20 and 18 KD expressed by both the clone and E. ictaluri cells. Genes encoding these proteins had no homology with other known genes. Overlapping inserts of clones 5d2 and 5d3 encoded homologs of E. coli partial genes serA and pgk, and complete genes rpiA, iciA, yggE, yggB and fda. Cloned antigenic E. ictaluri proteins of 33, 27, 35 and 45 KD were putatively identified as products of yggE, rpiA, iciA and fda respectively. Protective capabilities of vaccines of cloned antigenic proteins were evaluated in catfish. All vaccine treatments were protective against E. ictaluri challenge, however results were inconclusive due high levels of cross-reactive protection of E. coli, host strain of the cloning vector.

ISBN

9780591766721

Pages

252

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