In vitro cultivation of Pneumocystis isolated from infected rat lungs
Alicja Sobolewska1, Tadeusz H Dzbeński
1Department of Medical Parasitology, National Institute of Public Health-National Institute of Hygiene, Chocimska 24, 00-791 Warszawa, Poland. asobolewska@pzh.gov.pl
Abstract:
The studies were undertaken to check the possibility of a long-term in vitro cultivation of Pneumocystis obtained from immunosuppressed rats using slightly modified method of Merali et al. The growth of Pneumocystis in the established axenic cultures was examined by counting the number of cysts in Giemsa and Diff-Quik stained preparations or by estimating the number of DNA copies with a real-time PCR method. Growing organisms were subpassaged at 7-day intervals for at least 6 weeks, however, the highest growth of Pneumocystis was usually noted in the primary and the first 3 subcultures, reaching an average of 175-fold increase in the number of cysts and 286-fold increase in the number of DNA copies in primary cultures. The organisms collected from in vitro cultures were examined for immunogenic and antigenic properties showing the ability to raise high-titre antisera in rabbits. The immune sera proved very valuable in a Western-blot analysis of Pneumocystis antigens and in immunodiagnostic tests, such as dot-ELISA, enabling to detect circulating Pneumocystis antigens in bronchoalveolar lavage and serum samples from infected rats. Production of diagnostic antisera is so far the main advantage of the successful in vitro cultivation of Pneumocystis in axenic media.
Insights
Researchers successfully cultivated Pneumocystis in vitro for long-term study. This axenic culture method enables the production of diagnostic antisera for Pneumocystis infections.
Area of Science:
- Medical Mycology
- Infectious Diseases
- Parasitology
Background:
- Pneumocystis is an opportunistic pathogen causing pneumonia in immunocompromised individuals.
- Long-term in vitro cultivation of Pneumocystis is challenging but crucial for research.
Purpose of the Study:
- To establish a long-term in vitro cultivation method for Pneumocystis from immunosuppressed rats.
- To evaluate the growth kinetics and antigenic properties of cultivated Pneumocystis.
- To assess the utility of cultivated Pneumocystis for diagnostic antiserum production.
Main Methods:
- Modified Merali et al. method for axenic cultivation.
- Giemsa and Diff-Quik staining for cyst enumeration.
- Real-time PCR for DNA copy estimation.
- Subculturing at 7-day intervals for 6 weeks.
- Immunization of rabbits to produce antisera.
- Western-blot and dot-ELISA for antigen detection.
Main Results:
- Achieved sustained in vitro cultivation of Pneumocystis for at least 6 weeks.
- Observed highest growth in primary and first 3 subcultures (175-fold increase in cysts, 286-fold increase in DNA copies).
- Cultivated Pneumocystis demonstrated immunogenic and antigenic properties, raising high-titre antisera in rabbits.
- Generated immune sera valuable for Western-blot and dot-ELISA detection of Pneumocystis antigens in infected rat samples.
Conclusions:
- Successful long-term axenic cultivation of Pneumocystis is feasible.
- The established method supports Pneumocystis growth and maintains its antigenic properties.
- The primary advantage is the production of diagnostic antisera for Pneumocystis detection.
