Isolation of Edwardsiella ictaluri from channel catfish by tissue homogenization, filtration and enzyme linked immunosorbent assay

Abstract

An enzyme linked immunosorbent assay (ELISA) was developed to defect and quantify Edwardsiella ictaluri in asymptomatic carrier channel catfish Ictalurus punctatus and was compared to conventional culture on agar. Tissue homogenization, using 0.5% Triton X-100 in 0.05 M phosphate buffered saline (pH 7.2), clarified tissue slurries, improved filterability and permitted live bacteria to be filtered from 1 g or more of channel catfish organ tissue. Treatment of tissues with Tween 20 or trypsin did not allow improved filtration or bacterial counts. Bacteria captured on 0.45 μm nitrocellulose membrane filters were cultured on E. ictaluri medium agar. Bacterial colonies on the filters were tagged with a monoclonal antibody to E. ictaluri on which an ELISA was performed. The ELISA substrate 3,3'diaminobenzidine facilitated rapid bacterial colony forming unit (CFU) counts without magnification, permitted semipermanent records of isolations, and enabled microscopic confirmation following mounting with Permount. Assay sensitivity was less than 10 CFU g-1 of tissue. Conventional bacterial isolation and filter ELISA screening of 98 channel catfish from a population experiencing an outbreak of enteric septicemia of catfish (ESC) showed an injection prevalence of 24% by standard bacterial isolation techniques and 80% by filter ELISA in asymptomatic fish.