In Vitro Cultivation for Glugea plecoglossi (Microsporidia) Isolated from Ayu (Plecoglossus altivelis).

Glugea plecoglossi is an obligate intracellular microsporidium, which poses a significant threat to ayu (Plecoglossus altivelis). In vitro cultivation models are invaluable tools for investigating intracellular microorganisms, including G. plecoglossil. In this study, we attempted to in vitro cultivate G. plecoglossi 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 PaIL-1ß, PaTNF-α, PaIL-10, and PaTGF-ß, 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, G. plecoglossi spores recovered from EPC cells could infect young ayu, suggesting that EPC cells might be used as an in vitro cultivation system for G. plecoglossi.

Parallel detection of multiple zoonotic parasites using a real-time fluorogenic loop-mediated isothermal amplification-based quadruple-sample microfluidic chip.

Zoonotic parasites pose significant health risks globally. In the present study, we combined a microfluidic chip with loop-mediated isothermal amplification (on-chip LAMP) to detect five zoonotic parasites: Toxoplasma gondii, Cryptosporidium parvum, Cryptosporidium hominis, Clonorchis sinensis, and Taenia solium. This method enabled the simultaneous parallel analysis of five genetic markers from a maximum of four samples per chip. The on-chip LAMP assay was conducted in a highly automated format via the addition (by pipetting) of each sample in a single operation. The reaction was performed in volumes as low as 5 µL at a temperature of 65°C for 60 min, achieving limits of detection ranging from 10-2 to 10-3 pg./µL of recombinant plasmid DNA. All the time-to-positive values were less than 40 min, and almost all the coefficients of variation were less than 10%, even when using limit of detection concentrations for multiple pathogens, indicating robust reproducibility among replicates. The clinical sensitivity and specificity for detecting 135 field samples were 98.08 and 97.59%, respectively, compared with traditional biological methods, indicating good applicability in the detection of field samples. This on-chip LAMP assay allows for low reagent consumption, ease of operation, and multiple analyses of samples and genetic targets, and is applicable for on-site detection and the routine monitoring of multiple zoonotic parasites.