Liang WanWen

Development of a colloidal gold immunochromatographic strip for rapid detection of Streptococcus agalactiae in tilapia.

A colloidal gold immunochromatographic strip was developed for rapid detection of Streptococcus agalactiae (S. agalactiae) infection in tilapia. The monoclonal antibodies (mAb) 4C12 and 3A9 were used to target S. agalactiae as colloidal gold-mAb conjugate and captured antibody, respectively. The colloidal gold immunochromatographic strip was assembled via routine procedures. Optimal pH and minimum antibody levels in the reaction system for gold colloidal-mAb 4C12 conjugation were pH 7.4 and 18μg/mL, respectively. Optimal concentrations of the captured antibody 3A9 and goat anti-mouse antibody were 0.6mg/mL and 2mg/mL, respectively. The sensitivity of the strip for detecting S. agalactiae was 1.5×105 colony forming units (CFU). No cross-reaction was observed with other commonly encountered bacteria, including Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio anguillarum and Streptococcus iniae. The assay time for S. agalactiae was less than 15min. Tilapia samples artificially infected with S. agalactiae were tested using the newly developed strip. The results indicated that blood, brain, kidney, spleen, metanephros and intestine specimens of infected fish can be used for S. agalactiae detection. The validity of the strip was maintained for 6 months at 4°C. These findings suggested that the immunochromatographic strip was effective for spot and rapid detection of S. agalactiae infected tilapia.

Effects of recombinant tHsp70 on immune function of tilapia peritoneal macrophages

We used recombinant tilapia Hsp70(tHsp70) from Pichia yeast to prepare non-covalent HSP70-antigen complexes with thallus and extracellular bacterial products(ECP) of Streptococcus iniae.We investigated the ef-fects of the antigen complexes on the cellular immune response of tilapia by detection of NO release,phagocytic activity,and related immune gene expression of peritoneal macrophages in vitro.tHsp70 can form complexes with the thallus and ECP of tilapia in vitro.In addition,tHsp70 alone and its complexes significantly stimulated growth and attachment of peritoneal macrophages and enhanced NO release and phagocytic activity of peritoneal macro-phages.In contrast,treatment with ECP significantly inhibited NO release and led to cell death,while treatment with tHsp70-ECP complex reduced the cell death.Moreover,tHsp70 alone or tHsp70-antigen complexes signifi-cantly increased the expression of immune related genes(IL-8,Hsp70,CXCR4,and PGRN) in tilapia peritoneal macrophages compared to antigen only and medium control groups.Our results suggest that tHsp70 has an im-munoadjuvant and immunopotentiator function,and provides experimental data for development of a tHsp70 based vaccine against tilapia streptococcal diseases.