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Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection
Published on: September 14, 2019
An improved in vitro infection model for viability testing of Cryptosporidium parvum oocysts
Michael Najdrowski1, Anja Joachim, Arwid Daugschies
1Institute of Parasitology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 35, D-04103-Leipzig, Germany.
Abstract:
An in vitro infection model for the protozoan parasite Cryptosporidium parvum was evaluated for its suitability to determine the viability status of oocysts. Adherent HCT-8 cells were used as host cells and confluent monolayers were inoculated with oocyst suspensions in the presence of 0.4% sodium taurocholate which proved not to be cytotoxic. For a semi-quantitative detection of the infection a PCR-based assay was developed. The influence of physical (elevated temperature) and chemical (chlorocresole) inactivation methods on oocyst viability were evaluated. A minimum of 1000 untreated oocysts was necessary to establish a reproducibly detectable infection of the cells. With 10 and 100 oocysts, 30 and 78% of cell cultures, respectively, could be diagnosed as infected. For thermal inactivation two different temperature levels were used (38 and 55 degrees C). 55 degrees C, irrespective of incubation time, was sufficient to inactivate the oocysts to a degree below the detection limit. An elevation of temperature to 38 degrees C, in contrast, had no appreciable effect on oocyst infectivity in cell culture. Neopredisan efficacy against the parasite was tested at 0.25, 1 and 4% concentration. 0.25 and 1% had no discernible inhibiting effect on the developmental potential of the oocysts, while 4% Neopredisan resulted in a significant inhibition of Cryptosporidium development which was, however, not as prominent as heating to 55 degrees C, and not all oocysts could be inactivated.
Insights
An in vitro model using HCT-8 cells can assess Cryptosporidium parvum oocyst viability. High temperatures (55°C) effectively inactivate oocysts, while lower temperatures and certain disinfectants show limited efficacy.
Area of Science:
- Parasitology
- Cell Biology
- Infectious Diseases
Background:
- Cryptosporidium parvum is a significant protozoan parasite causing gastrointestinal illness.
- Assessing oocyst viability is crucial for understanding transmission and developing control strategies.
- Existing methods for viability assessment can be labor-intensive or lack sensitivity.
Purpose of the Study:
- To evaluate an in vitro infection model using HCT-8 cells for determining Cryptosporidium parvum oocyst viability.
- To assess the efficacy of physical (heat) and chemical inactivation methods on oocyst viability.
Main Methods:
- An in vitro model was established using adherent HCT-8 cells as host cells.
- Inoculation was performed with oocyst suspensions in the presence of non-cytotoxic sodium taurocholate.
- A semi-quantitative PCR-based assay was developed for infection detection.
- Oocysts were subjected to thermal inactivation (38°C and 55°C) and chemical treatment (Neopredisan).
Main Results:
- A minimum of 1000 untreated oocysts was required for reproducible infection detection.
- Infection was detected in 30% and 78% of cultures with 10 and 100 oocysts, respectively.
- Heating to 55°C effectively inactivated oocysts below the detection limit.
- 38°C had no significant effect on oocyst infectivity.
- 4% Neopredisan showed significant inhibition of parasite development, but was less effective than 55°C heat treatment.
Conclusions:
- The HCT-8 cell culture model provides a suitable platform for assessing Cryptosporidium parvum oocyst viability.
- Thermal inactivation at 55°C is a highly effective method for rendering oocysts non-infectious.
- Chemical disinfectants like Neopredisan require further optimization for complete oocyst inactivation.
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