This study demonstrates that a large 85 kb plasmid (pBLOOl) is essential for virulence of Salmonella pullorum, the causative agent of bacillary white diarrhea in chickens. Researchers used transposon mutagenesis to tag the plasmid and acridine orange-induced curing to eliminate it from strain 3, creating a plasmid-free derivative. The cured strain lost all virulence when administered orally to newly hatched chickens and showed significantly reduced virulence (12- to 150-fold increase in LD50) via intramuscular inoculation. Crucially, reintroducing pBLOOl via bacterial mating restored virulence in multiple independent transconjugants, fulfilling the criteria for causal association. Pathogenesis studies revealed that pBLOOl contributes to bacterial invasiveness through the alimentary tract and enables systemic spread to internal organs. The plasmid-containing strain persisted longer in liver, spleen, and heart muscle and produced characteristic cardiac lesions, whereas the plasmid-free strain failed to colonize these tissues effectively. Both strains grew equally well in vitro and were serum-resistant, indicating the plasmid's role is specific to pathogenic mechanisms rather than general survival.

Key findings

• An 85 kb plasmid (pBLOOl) is essential for S. pullorum virulence, as demonstrated by loss of virulence upon plasmid curing and restoration by plasmid reintroduction
• Plasmid loss completely prevented mortality from oral inoculation and increased intramuscular LD50 by 12- to 150-fold in newly hatched chickens
• pBLOOl mediates invasiveness through the alimentary tract and enables systemic spread to the reticuloendothelial system, as shown by reduced organ colonization in plasmid-free strains
• The plasmid-containing strain localized in high numbers in cardiac tissue and produced myocardial lesions typical of pullorum disease, unlike the plasmid-free derivative
• Plasmid presence correlates with earlier tissue invasion and prolonged persistence in liver and spleen following oral inoculation