Detection of cytotoxins produced by avian pathogenic E. coli and their cytotoxicity assay in CEF and VERO cells

Pathogenic strains of avian E.coli produces cytotoxins that induce morphological alterations in CEF and VERO cells. For the production of cytotoxins, avian E.coli isolates were grown in TSB medium and incubated in shaking water bath for 18–20 h. The cell free culture supernatants obtained after centrifugation (10000 rpm at 40C for 30 min) were filtered through 0.22μm membrane filter. The cytotoxicity assay was performed in 96 wells microtitre tissue culture plates. Out of 24 E.coli strains tested, 22(91.7%) exhibited cytotoxic affects on both CEF and VERO cells indicating production of enterotoxins whereas the toxins elaborated by 2(8.3%) serotypes produced a distinct cytotoxic response in VERO cells indicating production of verotoxin (VT). After heat treatment (85°C for 30 min) all the toxins gave no reaction on both monolayers except cytotoxic effect indicating that these were heat labile (LT) toxins and none of the isolates produced heat stable (ST) toxin. The titres of toxins assayed in terms of cytotoxic activity in CEF by various E.coli ranged from 1:4 to 1:1204, while in VERO cells it ranged from 1:4 to 1:64. However majority of the serotypes were having toxin titre of 1:16 in both the cell lines i.e. CEF and VERO.