Isolation and characterization of Salmonella Gallinarum cytotoxic factors

Two distinct cytotoxic factors isolated from a Salmonella Gallinarum strain recovered from a bird died during an outbreak of fowl typhoid were purified to homogeneity through ciprofloxacin extraction, salt precipitation, dialysis, gelfiltration, ionexchange chromatography and chromatofocusing. These were designated as Salmonella Gallinarum cytotoxin I (GCT-I) and II (GCT-II). GCT-I was a glycoprotein having mol.wt and pI of Ca 70 kDa and 8.8, respectively. It was lethal to birds (LD50, 150 micrograms) inducing fowl typhoid like lesions. GCT-II, a protein with Ca 55 kDa mol.wt., was not lethal but caused haemorrhagic diarrhoea on intraperitoneal inoculation in birds. Both the cytotoxins induced cytopathic effects (CPE) in Vero and Madin Darby bovine kidney (MDBK) cells, enterotoxicity in rabbit ileal loop, dermatotoxicity in the rabbit skin and specific neutralizing antibodies in rabbits. These were active only between a narrow pH range of 6 to 8.5 and thermostable at 90 degrees C (1 min) but lost their activities on boiling. Trypsin and chymotrypsin enhanced their cytotoxicity, while pepsin, papain, protease, lipase and urea (5 M) had no appreciable effect on their cytotoxicity. Sodium carbonate (0.05 M) and formaldehyde (0.05%) had no effect on antigenicity of both the cytotoxic factors but rendered them nontoxic. Identification and characterization of cytotoxic moieties of S. Gallinarum not only reveals the important virulence factor but also indicates about the use of toxic factors as a candidate for toxoid vaccine and immunodiagnostics.