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ISOLATION OF PURIFIED OOCYST WALLS AND SPOROCYSTS FROM TOXOPLASMA GONDII
Citation:
Everson, W. V., M W. Ware, AND H.D A. Lindquist. ISOLATION OF PURIFIED OOCYST WALLS AND SPOROCYSTS FROM TOXOPLASMA GONDII. JOURNAL OF EUKARYOTIC MICROBIOLOGY 49(4):344-349, (2002).
Impact/Purpose:
1) Refine new, practical methods for the detection of CCL-related and emerging waterborne human protozoa.
2) Perform field tests of devices or methods that have been developed under this task.
3) Evaluate these methods or devices in a variety of water matrices and parasite concentrations.
This work in this task supports CCL2 and 3 and is expected to be completed by 9/07.
Description:
Toxaplasma gondii is a parasitic protozoan that infects a wide range of vertebrates, including humans. This report describes methods that have been developed for separation of oocyst components starting with the mechanical fragmentation of oocysts. Use of iodixoanol gradients and centrifugation gave the highest yield of relatively pure fractions enriched in oocyst walls and intact sporocysts. Percoll gradient centrifugation also resulted in fractions highly enriched in intact sporocysts. Flow Cytometry with fluorescence activated cell sorting (FACS) was useful for analytical purposes. 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. These novel methods for isolation of oocyst and sporocysts provide materials that have utility in many applications. Oocyst walls may be used as an enriched starting material for development of methods to detect the organism in environmental samples, sporocysts as a starting material for analysis of excystation, and use of either fraction for proteomic analysis of components unique to this life cycle stage.