Invasion and Replication of Photobacterium damselae subsp. piscicida in Fish Cell Lines

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The objective of this study was to evaluate the ability of Photobacterium damselae subsp. piscicida to invade and replicate within different fish cell lines. Channel catfish ovary (CCO), fathead minnow (FHM), and epithelioma papillosum cyprini (EPC) cell lines were all susceptible to invasion and supported replication of P. damselae in an in vitro invasion assay in which extracellular growth was controlled with gentamicin. The number of bacteria recovered from EPC and CCO cells increased significantly after 6, 12, and 18 h, indicating that P. damselae replicated within those cells. There was also a significant increase in EPC cells at 3 h. Although the number of bacteria recovered from FHM cells increased slightly after 3 and 6 h, it declined at 12 and 18 h. The decline, however, was due to the early release of bacteria from FHM cells associated with a greater susceptibility to initial infection, which resulted in early cell lysis and subsequent exposure to gentamicin in the media. Using light and electron microscopy, we observed the invasion of bacteria as early as 30 min after infection. Intracellular bacteria were initially contained in small, close-fitting vacuoles that developed into large, clear, spacious vacuoles over time. There was no evidence of bacteria free in the cytoplasm. The intracellular location of P. damselae was confirmed by means of transmission electron microscopy with ruthenium red staining to discriminate between the extra- and intracellular spaces. This is the first report that provides evidence of intracellular replication of P. damselae in cell lines.

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Journal of Aquatic Animal Health

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