Comparison of Four Density Gradient Centrifugation Protocols for Purification of Toxoplasma gondii PRU Brain Cysts

Toxoplasma gondii is an obligate intracellular zoonotic protozoan that establishes persistent brain cysts in infected hosts, causing chronic infection and neuropathological damage. Efficient isolation and purification of brain cysts are essential for studying its biological characteristics and developing effective control strategies. The present study aimed to establish a reliable method for purifying brain cysts of the T. gondii PRU strain with high yield, viability, and practicality. Brain cysts harvested from experimentally infected ICR mice were purified using four different density gradient centrifugation methods: lymphocyte separation medium (LSM), Percoll, cesium chloride (CsCl), and sucrose. Purification efficiency was systematically evaluated, and the infection model was validated via brain histopathology. Cyst counts and purification yields were quantified, while cyst and bradyzoite viability were assessed using FDA/PI staining, trypan blue exclusion, and in vivo infectivity assays. Infected mice displayed the most severe clinical signs at 15 days post-infection (dpi), accompanied by significantly reduced body weight compared with uninfected controls (P < 0.05) and prominent perivascular inflammatory infiltration in the brain. Among the four purification methods, Percoll, CsCl, and sucrose gradients yielded significantly higher cyst numbers than LSM (P < 0.001), with no significant differences observed among the three gradient-based methods. Cysts purified by Percoll and LSM retained high viability and remained fully infectious in mice. Sucrose-purified cysts exhibited decreased viability, but their bradyzoites remained infective. In contrast, cysts and bradyzoites purified by CsCl were completely non-viable. These results clarify the advantages and limitations of each protocol and provide an optimized technical reference for T. gondii brain cyst research, supporting the development of toxoplasmosis prevention and control measures.