Expression and immunofluorescent localization of the Edwardsiella tarda EvpC protein in infected Pelteobagrus fulvidraco

Abstract

To determine the expression and localization of immunofluorescent secretory proteins encoded by Edwardsiella tarda (E. tarda) EvpC gene in tissues of experimentally infected yellow catfish Pelteobagrus fulvidraco (P. fulvidraco), an indirect immunofluorescent staining method was performed for detecting EvpC protein. A rabbit anti-EvpC polyclonal antibody, raised against a recombinant 6× His-EvpC fusion protein expressed in Escherichia coli, was prepared, purified, and used as the primary antibody. Sixty fish (50 ± 2.2 g) were intraperitoneally administered with E. tarda (infected group). Tissues were collected and prepared for indirect immunofluorescent staining at 4, 8, 12, 24, 48, 72, and 96 h post-inoculation. E. tarda EvpC encoding protein antigens could be detected in the liver, spleen, and kidney at 4 h after challenge, in the intestine at 12 h after challenge, in brain tissue 24 h after challenge, and in renal interstitium and liver tissue at 48 h. In addition, the intensity of positive staining of E. tarda EvpCp antigen in various tissues increased sharply from 8 to 96 h, peaking around 48–72 h, and subsequently decreasing steadily after 96 h, thus suggesting the expression levels of E. tarda EvpCp in systemic organs to be closely correlated with disease progression. The distribution pattern of E. tarda EvpCp was more prevalent in the spleen, liver, and kidney after infection, than in the intestine and gills. The present study may be useful not only for describing the characteristics of EvpCp expression and distribution in the chosen tissue of P. fulvidraco, but also for understanding pathogenesis by E. tarda and providing a basis for further functional analysis of the gene.