Intracellular survival of Renibacterium salmoninarum in trout mononuclear phagocytes

In vitro infection of primary cultures of leukocytes from kidneys of rainbow trout Oncorhynchus mykiss showed that Renibacterium salmoninarum, an obligate pathogen of salmonids, survived within the mononuclear phagocytes (MP). Transmission electron microscopy revealed that intracellular survival of the bacterium partially depended on its ability to move from the phagosome and into the cytoplasm. Formalin-killed R. salmoninarum also escaped into the cytoplasm, albeit at a slower rate and after sustaining greater cell wall damage, suggesting that the extracellular protein of the bacterium plays a role in intracellular survival. The durability of its cell wall enhanced survival within MP and significant bacterial losses occurred only after 96 h as the MP died and exposed the bacteria to antibiotics in the media. The bacteria appeared to maintain a slow rate of intracellular division, and dividing bacteria were seen in the micrographs through 240 h. Live R. salmoninarum were cytotoxic to MP; however, the MP persisted in culture and killed limited numbers of the bacterium. Adherence of the bacterium to the surfaces of lymphocytes and erythrocytes was also noted. An economical and time-saving method for observing and quantifying information obtained from transmission electron microscopy is described. The colony-forming units assay and cell viability counts provided additional information to support the data from electron microscopy.