| Summary: | <i>Glugea plecoglossi</i> is an obligate intracellular microsporidium, which poses a significant threat to ayu (<i>Plecoglossus altivelis</i>). In vitro cultivation models are invaluable tools for investigating intracellular microorganisms, including <i>G. plecoglossil</i>. In this study, we attempted to in vitro cultivate <i>G. plecoglossi</i> using primary cultures derived from ayu monocytes/macrophages (MO/MΦ), a murine-derived macrophage cell line RAW264.7, and the epithelioma papulosum cyprini (EPC) cell line. The results demonstrated that MO/MΦ infected with spores exhibited a pronounced immune response which was presented by rapidly high expression levels of inflammatory cytokines, such as <i>PaIL-1β</i>, <i>PaTNF-α</i>, <i>PaIL-10</i>, and <i>PaTGF-β</i>, and detached within 96 h post-infection (hpi). Infected RAW264.7 cells remained capable of stable passage yet exhibited cellular deformation with a decrease in intracellular spores occurring around 8 days post-infection (dpi). In contrast, EPC cells promised a substantial parasite population, and the cytokine expression levels returned to normal by 8 dpi. In addition, <i>G. plecoglossi</i> spores recovered from EPC cells could infect young ayu, suggesting that EPC cells might be used as an in vitro cultivation system for <i>G. plecoglossi</i>.
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