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Identification and Characterization of Infectious and Non-Infectious Sub-Populations of Encephalitizoon Intestinalis Spores Purified from in Vitro Cell Culture

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Abstract:Background: Encephalitizoon intestinalis spores were propagated in rabbit kidney (RK-13) cells and were purified using density gradient (Percoll [registered trademark]) centrifugation. Purified spores were enumeraged and aliquotted using flow cytometry with cell sorting for use in dose response assays. Flow cytometry analyses suggested a heterogeneous distribution of spore size, with two distinct subpopulations. This observation was confirmed in independent laboratories that enumerated separate lots of purified E. intestinalis spores.

Project Description and Results: Viability dye staining of sorted spores showed that the smaller subpopulation was propidium iodide (PI) - positive and 4',6-diamidino-2-phyenylindole diacetate (DAPI) - positive while the larger subpopulation was PI- and only stained DAPI+ following pretreatment with ethanol. Approximately 18-20% of any batch of purified E. intestinalis spores were of the size range of the larger subpopulation. This observation was confirmed by evaluating 12 batches of E. intestinalis spores produced and purified over a 12 month period. Spores of each subpopulation were subsequently infected into RK-13 cells using an optimized in vitro cell culture coverslip assay. The larger spores were infective in the cell culture assay, whereas the smaller spores were noninfectious in RK-13 cells. To confirm identity of the subpopulations, spore DNA was subjected to polymerase chain reactoin (PcR) assay using primers directed against the 16S rRNA or B-tubulin genes. PCR products were sequenced and compared to those of the E. intestinalis sequences for 16S rRNA or B-tubulin genes deposited in GenBank. Sequence data indicated that both the large and small spore populations possessed E. intestinalisB-tubulin and 16S rRNA genes, and were not derived from contaminants.

Significance: These data highlight the fact that not all E. intestinalis spores purified form cell culture are infective or viable. Furthermore, the large number (90-82%) of non-viable/non-infectious spores must be taken in to account when designing microsporidia spore disinfection studies. The number of non-viable/non-infectious spores in any given dose could potentially result in significant over-estimation (1.0 log10 or greater) of viability reduction. Researchers should be aware that there is inherent variability in cell culture spore production. Furthermore, the viability of a batch of E. intestinalis spores purified from RK-13 cells may be as low as 20% of the total number of spores.
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Citation:Marshall, M. M., B. Hoffman, J. Moffett, H. Jost, D. Polchert, M. W. Ware, G. Stubaum, and D. Battigelli. Identification and Characterization of Infectious and Non-Infectious Sub-Populations of Encephalitizoon Intestinalis Spores Purified from in Vitro Cell Culture. Presented at International Symposium on Waterborne Pathogens, Cascais, Lisbon, Portugal, September 22-25, 2002.
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Contact: Mary P. O'Bryant - (919)-541-4871 or obriant.mary@epa.gov
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Division: Microbiological & Chemical Exposure Assessment Division
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Branch: Biohazard Assessment Research Branch
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Product Type: Sympos/Conf
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Presented: 09/22/2002
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Related Entries:
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Bullet Item Detecting Ccl-Related, Emerging and Regulated Waterborne Human Protozoa for Exposure Assessment
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Last Updated on Monday, October 22, 2007
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