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Incorporation of exogenous uracil by Cryptosporidium parvum in vitro
S J Upton1, M Tilley, R R Mitschler
1Division of Biology, Kansas State University, Manhattan 66506.
Abstract:
Oocysts of Cryptosporidium parvum were used to infect Madin-Darby bovine kidney cells. Cultures were incubated in a reduced-oxygen atmosphere in candle jars or in a 5% CO2-95% air atmosphere. At 72 h, parasites were quantitated microscopically and found to be enhanced 5.5-fold in the reduced-oxygen atmosphere. Using candle jars, we then determined that C. parvum was amenable to [3H]uracil incorporation assays and easily quantitated with this method.
Insights
Reduced oxygen levels significantly enhance Cryptosporidium parvum growth in cell cultures. This finding supports using [3H]uracil incorporation assays for quantifying the parasite, improving research efficiency.
Area of Science:
- Parasitology
- Cell Biology
- Microbiology
Background:
- Cryptosporidium parvum is an opportunistic pathogen causing significant gastrointestinal illness.
- Understanding Cryptosporidium parvum's optimal growth conditions is crucial for developing effective control strategies.
- Current methods for quantifying Cryptosporidium parvum in vitro can be labor-intensive.
Purpose of the Study:
- To investigate the effect of reduced oxygen atmosphere on Cryptosporidium parvum development in Madin-Darby bovine kidney cells.
- To evaluate the suitability of [3H]uracil incorporation assays for quantifying Cryptosporidium parvum under optimized conditions.
Main Methods:
- Infection of Madin-Darby bovine kidney cells with Cryptosporidium parvum oocysts.
- Incubation of cultures in reduced-oxygen (candle jars) versus standard (5% CO2-95% air) atmospheres.
- Microscopic quantification of parasites at 72 hours post-infection.
- [3H]uracil incorporation assay for parasite quantitation.
Main Results:
- Parasite quantitation revealed a 5.5-fold enhancement of Cryptosporidium parvum in the reduced-oxygen atmosphere.
- The [3H]uracil incorporation assay proved effective for quantifying Cryptosporidium parvum.
- Candle jar incubation facilitated parasite quantification using this method.
Conclusions:
- A reduced-oxygen environment significantly promotes Cryptosporidium parvum replication in vitro.
- The [3H]uracil incorporation assay is a viable and efficient method for quantifying Cryptosporidium parvum growth.
- Optimized atmospheric conditions can enhance parasite yield for in vitro studies.