This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jenkins, M. B.
Right arrow Articles by Ghiorse, W. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jenkins, M. B.
Right arrow Articles by Ghiorse, W. C.
Agricola
Right arrow Articles by Jenkins, M. B.
Right arrow Articles by Ghiorse, W. C.

 Previous Article  |  Next Article 

Appl. Environ. Microbiol., Oct 1997, 3844-3850, Vol 63, No. 10
Copyright © 1997, American Society for Microbiology

Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts

MB Jenkins, LJ Anguish, DD Bowman, MJ Walker and WC Ghiorse
Division of Biological Sciences, Cornell University, Ithaca, New York 14853, USA. mbj1@cornell.edu

The ability to determine inactivation rates of Cryptosporidium parvum oocysts in environmental samples is critical for assessing the public health hazard of this gastrointestinal parasite in watersheds. We compared a dye permeability assay, which tests the differential uptake of the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) by the oocysts (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992), with an in vitro excystation assay, which tests their ability to excyst and, thus, their metabolic potential and potential for infectivity (J.B. Rose, H. Darbin, and C.P. Gerba, Water Sci. Technol. 20:271-276, 1988). Formaldehyde-fixed (killed) oocysts and untreated oocysts were permeabilized with sodium hypochlorite and subjected to both assays. The results of the dye permeability assays were the same, while the excystation assay showed that no excystation occurred in formaldehyde- fixed oocysts. This confirmed that oocyst wall permeability, rather than metabolic activity potential, was the basis of the dye permeability viability assessment. A previously developed protocol (L. J. Anguish and W. C. Ghiorse, Appl. Environ. Microbiol. 63:724-733, 1997) for determining viability of oocysts in soil and sediment was used to examine further the use of oocyst permeability status as an indicator of oocyst viability in fecal material stored at 4 degrees C and in water at various temperatures. Most of the oocysts in fresh calf feces were found to be impermeable to the fluorochromes. They were also capable of excystation, as indicated by the in vitro excystation assay, and were infective, as indicated by a standard mouse infectivity assay. The dye permeability assay further showed that an increase in the intermediate population of oocysts permeable to DAPI but not to PI occurred over time. There was also a steady population of oocysts permeable to both dyes. Further experiments with purified oocysts suspended in distilled water showed that the shift in oocyst populations from impermeable to partially permeable to fully permeable was accelerated at temperatures above 4 degrees C. This sequence of oocyst permeability changes was taken as an indicator of the oocyst inactivation pathway. Using the dye permeability results, inactivation rates of oocysts in two fecal pools stored in the dark at 4 degrees C for 410 and 259 days were estimated to be 0.0040 and 0.0056 oocyst day- 1, respectively. The excystation assay gave similar inactivation rates of 0.0046 and 0.0079 oocyst day-1. These results demonstrate the utility of the dye permeability assay as an indicator of potential viability and infectivity of oocysts, especially when combined with improved microscopic methods for detection of oocysts in soil, turbid water, and sediments.


This article has been cited by other articles:

  • Brescia, C. C., Griffin, S. M., Ware, M. W., Varughese, E. A., Egorov, A. I., Villegas, E. N. (2009). Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping of Viable Cryptosporidium Oocysts. Appl. Environ. Microbiol. 75: 6856-6863 [Abstract] [Full Text]  
  • Ollivett, T. L., Nydam, D. V., Bowman, D. D., Zambriski, J. A., Bellosa, M. L., Linden, T. C., Divers, T. J. (2009). Effect of nitazoxanide on cryptosporidiosis in experimentally infected neonatal dairy calves. J DAIRY SCI 92: 1643-1648 [Abstract] [Full Text]  
  • Peng, X., Murphy, T., Holden, N. M. (2008). Evaluation of the Effect of Temperature on the Die-Off Rate for Cryptosporidium parvum Oocysts in Water, Soils, and Feces. Appl. Environ. Microbiol. 74: 7101-7107 [Full Text]  
  • Collick, A. S., Fogarty, E. A., Ziegler, P. E., Walter, M. T., Bowman, D. D., Steenhuis, T. S. (2006). Survival of Cryptosporidium parvum Oocysts in Calf Housing Facilities in the New York City Watersheds.. J. Environ. Qual. 35: 680-687 [Abstract] [Full Text]  
  • Quilez, J., Sanchez-Acedo, C., Avendano, C., del Cacho, E., Lopez-Bernad, F. (2005). Efficacy of Two Peroxygen-Based Disinfectants for Inactivation of Cryptosporidium parvum Oocysts. Appl. Environ. Microbiol. 71: 2479-2483 [Abstract] [Full Text]  
  • Walker, M., Leddy, K., Hagar, E. (2001). Effects of Combined Water Potential and Temperature Stresses on Cryptosporidium parvum Oocysts. Appl. Environ. Microbiol. 67: 5526-5529 [Abstract] [Full Text]  
  • Simmons, O. D. III, Sobsey, M. D., Heaney, C. D., Schaefer, F. W. III, Francy, D. S. (2001). Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration. Appl. Environ. Microbiol. 67: 1123-1127 [Abstract] [Full Text]  
  • Jenkins, M. B., Walker, M. J., Bowman, D. D., Anthony, L. C., Ghiorse, W. C. (1999). Use of a Sentinel System for Field Measurements of Cryptosporidium parvum Oocyst Inactivation in Soil and Animal Waste. Appl. Environ. Microbiol. 65: 1998-2005 [Abstract] [Full Text]  
  • Widmer, G., Orbacz, E. A., Tzipori, S. (1999). beta -Tubulin mRNA as a Marker of Cryptosporidium parvum Oocyst Viability. Appl. Environ. Microbiol. 65: 1584-1588 [Abstract] [Full Text]  
  • Robertson, L. J., Campbell, A. T., Smith;, H. V., Jenkins, M. B., Ghiorse, W. C., Anguish, L. J., Bowman, D. D., Walker, M. J. (1998). Viability of Cryptosporidium parvum Oocysts: Assessment by the Dye Permeability Assay. Appl. Environ. Microbiol. 64: 3544-3545 [Full Text]  
  • Walker, M. J., Montemagno, C., Bryant, J. C., Ghiorse, W. C. (1998). Method Detection Limits of PCR and Immunofluorescence Assay for Cryptosporidium parvum in Soil. Appl. Environ. Microbiol. 64: 2281-2283 [Abstract] [Full Text]  
  • Kaucner, C., Stinear, T. (1998). Sensitive and Rapid Detection of Viable Giardia Cysts and Cryptosporidium parvum Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR. Appl. Environ. Microbiol. 64: 1743-1749 [Abstract] [Full Text]  
  • Jenkins, M. B., Bowman, D. D., Ghiorse, W. C. (1998). Inactivation of Cryptosporidium parvum Oocysts by Ammonia. Appl. Environ. Microbiol. 64: 784-788 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»