A Comparative Study of the Immunological Properties of Extracellular Products Between Virulent and Less Virulent Edwardsiella tarda

Abstract

The immunological properties of the extracellular products (ECP) of the Edwardsiella tarda parent FL6-60 and mutant RET-04 isolates were investigated in Nile tilapia Oreochromis niloticus. Non-purified ECP derived from the FL6-60 parent and RET-04 mutant stimulated in vitro migration of macrophages and the movement was demonstrated to be predominantly chemokinetic. Semi-purification of the ECP by high pressure liquid chromatography (HPLC) identified components with molecular weights of 31.62, 3.19, and 0.60 kDa for the FL6-60 parent, and 5.65, 0.55, and 0.10 kDa for the RET-04 mutant ECP. Analysis of chemoattractant activity for both the semi-purified FL6-60 parent and RET-04 mutant ECP revealed primarily chemotactic migration and macrophage chemotaxis was increased over the non-purified fractions. In assays of both non-purified and semi-purified ECP fractions, differences between macrophage movement induced by the FL6-60 parent and RET-04 mutant ECP were minimal. Additionally, protective immunity conferred by passive immunization was investigated. Hyperimmune serum was obtained from fish actively exposed to and challenged with E. tarda FL6-60, while nonimmune serum was acquired from fish IP administered tryptic soy broth (TSB). Fish were passively immunized by IP injection with pooled hyperimmune or nonimmune serum, or phosphate buffered saline (PBS), as a control, and sera were collected at 72 h after immunization. Antibody titer was significantly (P <0.0001) higher in fish obtained from the hyperimmune group (1:25585) than in the nonimmune (1:606) or PBS (1:399) groups. Mean cumulative percent mortality was significantly (P <0.0001) lower in the hyperimmune group (6.7%) than in the nonimmune (26.7%) or PBS (17.9%) groups. Immunization by intraperitoneal (IP) injection of FL6-60 parent and RET-04 mutant ECP was also studied. Following challenge, differences in cumulative percent mortality of the FL6-60 parent (64.6%), and RET-04 mutant (63.8%) ECP immunized groups, and TSB control group (79.5%) were not significant (P <0.3541). Characterization of the FL6-60 parent and RET-04 mutant ECP by electrophoretic analysis revealed differences in banding profiles between the FL6-60 parent and RET-04 mutant ECP, most noticeably with a band of approximately 175 kDa resolved in the RET-04 mutant ECP absent in the FL6-60 parent ECP.