CHARACTERIZATION OF PNEUMOCYSTIS-CARINII PREPARATIONS DEVELOPED FOR LIPID ANALYSIS

Pneumocystis carinii organisms were isolated from viral antibody-negat ive rats that had been infected by intratracheal intubation of organis m preparations tested negative for common bacteria and fungi. Infectio n scores of lungs from infected animals at the time of parasite isolat ion was >5(100-1,000 organisms/oil immersion field). Electron microsco py of heavily infected lungs revealed that the pathogens adhered to Ty pe 1 pneumocytes and to each other, resulting in obstructions up to se veral cell layers thick, which extended into the alveolar lumen. Proto cols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation puri ty, recovery efficiency, and organism viability. The study tested muco lytic agents, sieving, various centrifugation speeds, lysis of host ce lls by osmotic shock and filtration through membranes of different por e diameter. Final preparations contained no intact host cells as deter mined by light microscopy. Only minor amounts (<5%) of host debris wer e detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. Th e final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indic ated >99.5% purity. The total non-P. carinii protein in the final prep aration (<6%, depending on the level of infection) was estimated by th e protein content of pelletable material resulting from processing uni nfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purif ication process. Exogenous stigmasterol, added as an extracellular mar ker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 10(8)-10(9) organisms/rat w ere routinely obtained. Viability, assessed by the calcein acetoxymeth yl ester-propidium iodide assay, was 80-95%.