Figure 1.
Growth of sorted P. carinii organisms in axenic culture.
Several P. carinii populations were cultured in DMEM with 10% FBS, at 37°C, in an atmosphere containing 5% CO2. Growth of (A) sorted P. carinii total population devoid of host cell debris, (B) pure P. carinii trophic forms, and (C) pure P. carinii cystic forms is followed during 4 days. Trophic forms (circles, left Y-axis) or cystic forms (triangles, right Y-axis) were microscopically quantified after RAL-555 panoptic staining [26-28]. For each population of P. carinii organisms studied, means of three replicates are represented per time point. Error bars represent standard deviations. The star (*) means P-value ≤ 0.05. The detection limit is 10 P. carinii organisms per culture well.
Figure 2.
Infection of nude rats with sorted P. carinii organisms.
Nude rats were endotracheally infected with three P. carinii sorted populations: (A) sorted P. carinii total population devoid of host cell debris, (B) pure P. carinii trophic forms, and (C) pure P. carinii cystic forms. Rats were euthanized at 0.5 (12 h), 2.5 days, and 8.5 days postinoculation and then parasites were extracted from lungs [29]. After staining with RAL-555, trophic forms (in grey) or cystic forms (in black) were microscopically quantified [26-28]. At each time point, means of either trophic- or cystic-form burdens developing in the lungs of three animals are plotted on the same Y-axis. Error bars represent standard deviations. The star (*) means P-value ≤ 0.05.
Figure 3.
Purity of sorted P. carinii cystic and trophic forms.
P. carinii cyst forms were physically separated from trophic forms by cell sorting using high-speed flow cytometry [12]. To check for purity, smears of sorted P. carinii organisms were stained with RAL-555, a panoptic Giemsa-like stain. No cystic forms were noted in the sorted trophic-form fraction (A). No trophic forms were visible within the sorted cystic-form fraction (B). P. carinii cell sorting was reproducible, reaching 99.6% purity [12]. Insets represent a higher magnification. Bars = 10 μm.
Figure 4.
Nude rat model of aerial P. carinii transmission.
To study the airborne transmission of P. carinii, three groups of animals were infected with either P. carinii total population or trophic or cystic forms. To ease comprehension, only a portion (1/4) of the animals belonging to a single group is represented on the figure. First, seeder rats (in grey) were endotracheally infected with P. carinii organisms (total sorted P. carinii population, pure trophic forms, or cystic forms) [23]. Second, after 15 min of recovery, seeder rats were placed in close contact with receiver animals (in white) for 12 h at a mean ratio (seeders to receivers) of 2.4 per cage. These dexamethasone-treated animals were co-housed in capped cages within an individual compartment (thick black square line) of an HEPA-filtered air isolator. Third, all the seeder and half of the receiver rats were euthanized at the end of the contact period. P. carinii organisms were extracted from the seeder rat lungs [29]: the fungal burden was microscopically quantified [26-28] and molecular detection of the P. carinii mtLSUrRNA gene was performed using a single-round touchdown PCR (TD-PCR, [32]). Whole lung tissue suspensions of half of the receiver rats (dotted square line) were screened by nested-PCR (nPCR) at the same locus to detect P. carinii gDNA [5]. Fourth, the other half of the receiver rats (dotted oval line) were kept under immunosuppression (IS) in HEPA-filtered air conditions for 7 weeks to microscopically monitor for eventual PcP development. nPCR was also performed on whole lung tissue suspensions to detect P. carinii gDNA. Whole lung tissue suspensions of sentinel rats were also screened by nPCR and by microscopy after 7 weeks of immunosuppression.