Studies on Rapid Diagnosis System of Main Bacterial Diseases of Pond-cultured Eels in Japan-I

Basic Investigations on the Diagnosis of Edwardsiellosis by Direct Immunofluorescence

Abstract

Epizootics of edwardsiellosis has been frequently occurring among pond-cultured eels, Anguilla japonica in Japan. Accurate diagnosis of the disease by means of conventional cultural method requires a day or more for completion. In this report, basic investigations for the application of direct immunofluorescence as rapid diagnostic tools of the disease were made.
Antiserum was obtained from rabbit immunized with formalin-killed E. tarda (strain E-8) isolated from eel. The titer against formalin-killed homologous antigen was 1: 2048 by agglutination test. IgG of the antiserum was fractionated by ammonium sulfate precipitation and conjugated with FITC. The F/P molar ratio of labeled antibody was determined as 1.3.
The labeled antibody had staining titer of 1:16 or 1:32 against 18 strains of E. tarda, including homologous strain E-8, isolated from pond-cultured eels at various regions in Japan. But no crossreaction was observed when 2 strains of V. anguillarum, 3 of Vibrio sp., 3 of A. liquefaciens, 8 of Aeromonas sp., one of Ps. anguilliseptica, one of Ps. fluorescens, one of Ps. putida, one of Escherichia coli, 2 of Salmonella sp., one of S. marcescens and one of F. columnaris were stained.
The specific fluorescence of target antigen (E. tarda) was observed when acetone-fixed impression preparations of liver, kidney, spleen and injection site of muscle derived from artificially infected eel were stained with 4-fold dilution of labeled antibody. The specificity of the reaction on impression preparation was ascertained by one-step inhibition test. Stainability of E. tarda on impression preparation was stable for at least 11 days at 37°C. Stainability of E. tarda on impression preparation of liver tissue that had been preserved in 10% formalin for at least 7 days, though became slightly weaker, was not disappeared.