[Continuous axenic cultivation of Pneumocystis carinii isolated from the bronchoalveolar lavage fluid of infected rat]

Abstract

Objective: To establish axenic cultivation of Pneumocystis carinii (P.c).

Methods: The organisms of P.c were isolated from the bronchoalveolar lavage fluid (BALF) of the rats with Pneumocystis carinii pneumonia (PCP) and cultured in a medium which was based on IMDM(GIBCO) supplemented with S-adenosyl-L-methionine, putrescine, N-acetyl glucosamine, putrescine, L-cysteine and L-glutamine, and newborn calf serum. The organisms cultured in the system were identified by observing the morphology of cysts in smears stained with Gomori's methenamine silver nitrate stain (GMS). Ultrastructure of the cysts/trophozoites was examined by transmission electron microscopy. The sequences of mitochondrial large ribosomal DNA subunit of the cultured organisms were compared with the Pneumocystis carinii f.sp. ratti variant isolate (GenBank No U20173) and Pneumocystis carinii f.sp.hominis (GenBank No M58605).

Results: Five isolates of P. carinii received from BALF of 8 rats with PCP were cultured axenically and continuously in the system. The cultured organisms could be stored in frozen condition and used to reinitiate culture, and were amplified by 19-22 times within 72 h. The morphology, ultrastructure and gene sequencing of the cultured organisms confirmed that the isolated organisms were P. carinii.

Conclusion: Five continuously and axenicly cultured isolates of P. carinii have been received.