In vitro cultivation of Pneumocystis isolated from infected rat lungs.

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.