Purification of Kudoa septempunctata myxospores by using trypsin and fluorescence activated cell sorting

Kudoa septempunctata, a myxozoan, has been identified as the causative agent of foodborne illnesses associated with the consumption of raw olive flounder. To develop effective control methods against this parasite, fundamental research-including viability determination, transcriptome analysis, and antigenicity assessment of K. septempunctata myxospores-is required. This research necessitates the purification of the parasites. Sequential trypsin digestion, followed by density gradient purification, was performed to isolate the K. septempunctata myxospores. Further purification was achieved through fluorescence-activated cell sorting at a concentration of 106 to 107 myxospores/ml. The results demonstrated that the combination of trypsin digestion and density gradient methods consistently produced approximately 40 times more viable myxospores than the density gradient method alone. Additionally, the fluorescence-activated cell sorting method enhanced the purity of the myxospores by approximately 10%. The procedures described in this study will support research (such as RNA-sequencing, proteomics, vaccine antigen preparation) aimed at developing control methods for K. septempunctata including the fundamental research.