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Twenty-four strains of Salmonella enteritidis, isolated from several outbreaks of salmonellosis from different poultry farms in India, were checked for the plasmid profile and detection of virulence gene(s) by PCR. Most of the strains contained only a single plasmid of 55 kb. Additional plasmids of 23.2 kb and 8.7 kb were seen in one of the strains, and another strain carried only two plasmids of 23.2 kb and 8.7 kb. Four strains did not carry any plasmid. PCR amplification showed the presence of virulence-associated genes in all the isolates harbouring the 55 kb plasmid. Intraperitoneal inoculation of mice, with most of the strains carrying the 55 kb plasmid, caused 100% mortality. Most strains lacking the 55 kb plasmid were avirulent. In chickens, oral inoculation of the S. enteritidis strains carrying the 55 kb plasmid produced 40–100% mortality, with characteristic signs of salmonellosis. Oral inoculation of strains lacking the 55 kb plasmid did not cause any mortality. Hence, it appears that the large plasmid of S. enteritidis probably contributes towards virulence in mice and chickens.