SURVIVAL AND REPLICATION OF RHODOCOCCUS-EQUI IN MACROPHAGES

Rhodococcus equi is a facultative intracellular bacterium of macrophag es that can cause serious pneumonia in both young horses and immunocom promised people. Essential to understanding rhodococcus pathogenesis i s a quantitative documentation of the intracellular events that follow macrophage phagocytosis of the organism. By using a bacterial immunof luorescence staining assay, we verified the intracellular survival and replicative potential of R. equi in both murine peritoneal macrophage s and equine alveolar macrophages in vitro. Following an initial lag p eriod of 6 to 12 h, the intracellular numbers of R. equi begin to rise , often reaching macrophage-compromising levels by 48 h. A quantitativ e determination of bacterial growth bg a novel image analysis cytometr y technique confirmed our fluorescence microscopic results. By 48 h po stinfection, bacterial numbers had increased by more than fivefold, an d the majority of infected macrophages in the monolayer contained 10 o r more bacteria per cell. The intracellular organisms were viable, as evidenced by the ability to incorporate radiolabeled uracil. The use o f these techniques has identified differences in the in vitro replicat ive capacities of a virulent strain and an avirulent strain of R. equi . A clinical isolate of R. equi ex-pressing a 17-kDa virulence-associa ted plasmid encoded antigen was able to survive and replicate within m acrophages, whereas an avirulent, non-plasmid-containing strain replic ated poorly. These results suggest that plasmid-encoded bacterial viru lence factors may contribute to the ability of R. equi to replicate wi thin its host cell, the macrophage.