Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii.
Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.
Everson
William V
WV
Microbiological and Chemical Exposure Assessment Research Division, United States Environmental Protection Agency, Cincinnati, Ohio 45268, USA.
Ware
Michael W
MW
Dubey
J P
JP
Lindquist
H D Alan
HD
D000818:Animals; D002415:Cats; D002473:Cell Wall / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D005434:Flow Cytometry; D005898:Glass; D008018:Life Cycle Stages; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010078:Oxazines; D010274:Parasitology / Q000379:methods*; D011205:Povidone; D012822:Silicon Dioxide; D014122:Toxoplasma / Q000254:growth & development* / Q000302:isolation & purification*; D014283:Triiodobenzoic Acids; D014732:Vibration
D010078:Oxazines; D014283:Triiodobenzoic Acids; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone; C044834:iodixanol; C044808:nile red