In vitro model to estimate Edwardsiella tarda-macrophage interactions using RAW264.7 cells

Edwardsiella tarda has been recognized as an important facultative intracellular pathogen of fish with capability of survival and replication within macrophages. E. tarda-macrophage interactions play a very important role in the defense mechanism of fish against infection. The mechanisms that E. tarda use to infect and persist inside macrophages are not well characterized. To gain insight concerning this process, RAW264.7 cells was used to investigate the interactions between E. tarda and macrophages. Using an in vitro model involving RAW264.7 cells, internalization assay demonstrated that MOIs of 10:1 and 100:1 could result in a satisfactory infection rate after a 2 h infection period. Consistent with the performance in fish macrophages, E. tarda could survive, replicate and induce iNOS-mediated NO production in RAW264.7 cells. Light and electron microscopy confirmed the internalization and replication of E. tarda in RAW264.7 cells, showing once inside macrophages, numberous bacteria may be destroyed within phagolysosomes and those that successfully subvert phagocyte defenses are capable of extensively replicating within the vacuolar-like compartment in macrophages. In addition, E. tarda-induced apoptosis was observed in RAW264.7 cells in a dose-and time-dependent manner, characterized by increased Annexin V binding and the activation of caspase-3. The results described here indicate that RAW264.7 cells could model the behavior of fish macrophages in response to E. tarda in many ways and may serve as a cell model for study on interactions between E. tarda and macrophages. The successful establishment of E. tarda-invaded RAW264.7 cells model may contribute to providing a basis for more detailed understanding of E. tarda pathogenesis.