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Updated: Oct 19, 2026
Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection
Published on: September 14, 2019
A most-probable-number assay for enumeration of infectious Cryptosporidium parvum oocysts
T R Slifko1, D E Huffman, J B Rose
1Department of Marine Science, University of South Florida, St. Petersburg, Florida 33701-5016, USA. slifko@seas.marine.usf.edu
Abstract:
Cryptosporidium is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20). A two-tailed P value (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log(10) inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.
Insights
A new cell culture assay accurately quantifies infectious Cryptosporidium oocysts in water. This focus detection method-most probable number (FDM-MPN) assay is valuable for water quality testing and disinfection studies.
Area of Science:
- Environmental microbiology
- Parasitology
- Water quality analysis
Background:
- Cryptosporidium oocysts are persistent contaminants in drinking and recreational water sources worldwide.
- Accurate quantification of infectious oocysts is crucial for assessing water safety and disinfection efficacy.
Purpose of the Study:
- To adapt and validate a cell culture infectivity assay for quantifying viable Cryptosporidium oocysts.
- To establish a reliable method for enumerating infectious oocysts in water samples.
Main Methods:
- A focus detection method-most probable number (FDM-MPN) assay was developed using HCT-8 cell monolayers.
- Infectious oocysts were quantified by sporozoite invasion and focus clustering.
- Oocyst infectivity was assessed in relation to age and pulsed white light treatment.
Main Results:
- The FDM-MPN assay demonstrated a strong correlation (r=0.9726) between calculated and inoculated infectious oocysts for young oocysts.
- The assay effectively enumerated viable oocysts, with high statistical significance (P < 0.0001).
- Both FDM-MPN and animal infectivity assays showed >4 log(10) inactivation after pulsed white light treatment.
Conclusions:
- The FDM-MPN assay provides a reliable method for enumerating infectious Cryptosporidium oocysts in cell culture.
- This assay can be utilized by municipal water systems and water testing organizations for routine survival and disinfection studies.
- Oocyst age significantly impacts infectivity, a factor the FDM-MPN assay can account for.
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