Early Acquisition of Pneumocystis carinii in Neonatal Rats using Targeted PCR and Oral Swabs

Rat groups

Ten rat dams and their pups were used for these studies, including 8 sets of Long Evans and 2 sets of Brown Norway rats, for a total of 121 rats. All rats used were bred and housed at the VA Medical Center Animal Facility, (rooms 003/004, Cincinnati, OH). Breeder rats and neonates were housed in open shoebox cages in a room with other rats known to harbor Pneumocystis carinii f. sp. carinii. Rats were handled according to IACUC guidelines under VA Medical Center protocols.

Oral swabs

Seven dams and their pups were analyzed for Pneumocystis exposure by the presence of Pneumocystis-specific DNA in the oral cavity, as described previously [18]. Oral swabs were collected from four of these rat dams and their pups (n=31) at 1, 2, or 4 weeks after birth using sterile cotton-tipped wooden applicators (Fisher, Pittsburgh, PA). Swabs were also collected from three rat dams and their pups (n=39) at 2, 24, 48, and 72 hours after birth using tapered mini sterile cotton tipped applicators (Hardwood Production Company LP, Guilford, ME). All oral swab samples were collected by rubbing a cotton swab over the hard palette, surface of the tongue, buccal surface and under the tongue of each rat. Each intact swab was vortexed in 200-500μl DNA extraction buffer (100mM Tris, 100mM EDTA, 200mM NaCl, 1% Sarkosyl). then the DNA was extracted using Phase Lock Heavy Gel tubes (Eppendorf, Westbury, NY). DNA samples were stored in TE (1mM EDTA, 10mM Tris-Cl) at -20°C until needed for PCR analysis.

Cesarean sections

Three gravid female Long Evans rats were chosen for cesarean sections. At 2 to 4 weeks gestation, oral swabs were collected and each rat was sacrificed by injection of approximately 500mg chloral hydrate (Major Pharmaceuticals, Livonia, MI). The intact uterus was removed from the abdominal cavity in a bioflow chamber (The Germfree Laboratories, Inc., Miami, FL) and placed in a sterile petri plate. The lungs, liver, and spleen were removed from each dam and placed in individual sterile tubes. For the gravid females, each pup was removed from the uterus, the placenta (including the deciduae) was removed from the pups, and each pup was placed in a separate sterile tube. The uterus and individual placentas were also placed in separate sterile tubes. All samples were homogenized in a proteinase K solution (0.5mg/ml extraction buffer; Roche, Indianapolis, IN) and digested over night at 55°C. DNA was extracted from 250 μl of each sample by a standard organic phase method and stored in TE at –20°C until analyzed by PCR.

PCR analysis

DNA samples from all oral swabs and tissue homogenates were amplified using the Rcc primer set [22] with a GeneAmp PCR System 9700 (PE Applied Biosystems, Norwalk, CT). The Rcc primers target a region of the mitochondrial large subunit rRNA (mtLSU rRNA) specific for P. carinii f. sp. carinii. Each reaction used 1 X JumpStart REDTaq ReadyMix PCR Reaction Mix (Sigma, St. Louis, MO) (10mM Tris-HCl, 2mM MgCl₂, 50mM KC1, 0.001 % gelatin, 0.2mM each dNTP, 0.06 unit/μ1 Taq DNA Polymerase, TaqStart antibody) to which 0.05ng each of Rcc1 and Rcc2 primers, 1.0 μl template DNA, and molecular grade water were added. PCR conditions were: 94°C hot start for 2 minutes, 94°C denaturing for 30 seconds, 54°C annealing for 30 seconds, 72°C extension for 60 seconds (40 cycles total), and 72°C final extension for 10 minutes. Amplified DNA products were visualized by staining with ethidium bromide in 2% agarose gels run at 90 V for 1 hour. Gel images were captured with NIH Image 1.6 software. Each rat was scored positive for P. carinii f. sp. carinii by the presence of a band at 137bp.

Oral swab results from rat pups within 4 weeks of age

Rat pups were sampled by the oral swab/PCR technique at ages ranging from 1 to 4 weeks (8 pups at 1 week, 12 pups at 2 weeks, 11 pups at 4 weeks). All 31 samples were positive for Pcc-specific DNA in the oral cavity (Table I). The 4 dams of these pups were also positive for Pcc-specific DNA by oral swab. Although the presence of Pec-specific DNA in the oral cavity of these pups does not prove that intact, viable Pneumocystis organisms were there, it does suggest that an exposure occurred.

Oral swab samples from rat neonates within 1 week of age

To better define the time of acquisition, a second set of oral swabs were collected from three rat dams and their pups at 1–2 hours, 24 hours, and 1 week after birth. 78% (28/36) of these pups were positive for Pec-specific DNA by oral swab within the first 2 hours of life (Table 1). By 24 hours and at 1 week of age, 100% were positive for Pec-specific DNA. The three dams were also positive for Pec-specific DNA by oral swabs during all sample time points. These data suggest that the pups sampled in this investigation were exposed to Pneumocystis within the first 2 hours of life, supporting the concept of early Pneumocystis acquisition.

Vertical transmission of Pneumocystis

Because rat pups were shown to be exposed to Pcc within the first 2 hours of life, the potential for transplacental transport was investigated. Rat pups were collected by cesarean section at three timepoints, ranging from 10 to 28 days gestation. None of the 48 rat fetuses, placentas or deciduae were positive for Pcc-specific DNA. In contrast, the three dams were positive for Pcc-specific DNA by oral swab, lung tissue homogenate, and spleen homogenate (Table 1).

In several earlier reports, investigators suggested that Pneumocystis could be transmitted from mother to pup by transplacental transport in humans, rats, and rabbits [8, 11, 17, 21, 25]. These observations were supported by numerous human and rat reports of extrapulmonary dissemination of Pneumocystis, as identified by histological examination of post-mortem tissues [1–4, 6, 7, 10, 15, 16, 20, 26, 31]. The direct correlation between Pneumocystis infection of neonates and transmission through the placenta has been difficult to verify. Our data indicate that the transplacental transport of Pneumocystis is not a mode of transmission in rats, supporting previous observations in SCID mice [19]. The close contact between the dam and pup immediately after birth would allow Pneumocystis to be transmitted from mother to pup, or Pneumocystis could be acquired from the immediate environment (i.e. bedding, feces, air).

These data provide a scientific rationale for the development of Pneumocystis-free rat colonies through cesarean section-derived rats; further validate oral swab/PCR as a tool for determining Pneumocystis exposure in rats; and support previous rat and human studies that report early exposure to Pneumocystis.