The association between a large molecular mass plasmid and virulence in a strain of Salmonella pullorum

Eight strains of Salmonella pullorum isolated from epidemiologically independent cases of pullorum disease (bacillary white diarrhoea) in young chickens possessed at least one large molecular mass plasmid in addition to smaller molecular mass plasmids. The 85 kb large plasmid, designated pBL001, of one of these strains was 'tagged' with an ampicillin resistance marker by the insertion of transposon Tn3. The plasmid was eliminated by passage in nutrient broth containing acridine orange. It was reintroduced into the strain from which it had been eliminated by mobilization using the F plasmid. Following oral inoculation of newly hatched Rhode Island Red chickens, the parent strain produced a high level of mortality (71%) with characteristic signs of pullorum disease. Following intramuscular inoculation of chickens of the same age, the bacterial LD50 was (log10 c.f.u.) 3.38 +/- 0.43 (mean +/- SEM). The derivative lacking pBL001 produced no mortality or morbidity when inoculated orally and the bacterial LD50 value increased to (log10 c.f.u.) 5.54 +/- 0.28. This increase was statistically significant (chi 2 = 13.6, P less than 0.01). Reintroduction of pBL001 restored virulence as gauged by oral inoculation of chickens (62% mortality) and by the intramuscular bacterial LD50 value (log10 c.f.u. = 3.78 +/- 0.25). These values were not significantly different to those produced by the parent strain (chi 2 = 0.59, P = 0.4 and chi 2 = 0.66, P = 0.5, respectively). Following oral inoculation, the pBL001-cured derivative was less invasive than the parent strain and following intramuscular inoculation it persisted for a shorter period than the parent strain in the liver, spleen and the leg muscle into which it had been inoculated. In addition, the parent strain, but not the pBL001-cured derivative, localized in large numbers in the myocardium where it produced lesions typical of pullorum disease. Both the parent strain and the pBL001-cured derivative were serum resistant in the presence of rabbit serum and grew equally well in chick serum and broth.