Widespread occurrence of Pneumocystis carinii in commercial rat colonies detected using targeted PCR and oral swabs

Abstract

The genus Pneumocystis contains a family of fungal organisms that infect a wide variety of mammalian species. Although it is a cause of pneumonia in immunocompromised hosts, recent evidence suggests that these organisms colonize nonimmunosuppressed hosts. Detection of cryptic colonization with Pneumocystis becomes important in animal studies when infection-free animals are necessary. Provocation by chronic immunosuppression, histology, and serology has been widely used to detect the presence of Pneumocystis in rat colonies, requiring lengthy time periods and/or postmortem tissue. We conducted a study to evaluate the use of PCR amplification of oral swabs for the antemortem detection of Pneumocystis in 12 rat groups from three commercial vendors. Sera were collected upon arrival, and the oral cavity was swabbed for PCR analysis. Ten of these groups of rats were then housed in pairs under barrier and immunosuppressed to provoke Pneumocystis growth. Once moribund, the rats were sacrificed, and the lungs were collected to evaluate the presence of Pneumocystis by PCR and microscopic enumeration. DNA was extracted from oral swabs and lung homogenates, and PCR was performed using primers targeting a region within the mitochondrial large-subunit rRNA of Pneumocystis carinii f. sp. carinii. Upon receipt, 64% of rats were positive for P. carinii f. sp. carinii-specific antibodies, while P. carinii f. sp. carinii DNA was amplified from 98% of oral swabs. Postmortem PCR analysis of individual lungs revealed P. carinii f. sp. carinii DNA in all rat lungs, illustrating widespread occurrence of Pneumocystis in commercial rat colonies. Thus, oral swab/PCR is a rapid, nonlethal, and sensitive method for the assessment of Pneumocystis exposure.

FIG. 1

Immunoblotting results for Hollister (area 42) serum samples. Lane 1 is the negative control (Tween-Tris-buffered saline [0.02 M Tris, 0.5 M NaCl, 0.05% Tween 20]), lane 2 is the positive control (Pneumocystis antibody-positive rat serum), and lanes 3 to 13 are Hollister area 42 rat sera. All rats have an antibody reaction with the 120- to 140-kDa MSG of P. carinii f. sp. carinii form 1 surface antigen preparation.

FIG. 2

PCR products showing the presence or absence of P. carinii f. sp. carinii DNA in nonimmunosuppressed Raleigh R09 rats. Lanes 1 to 12, individual rat samples; lane 13, a positive control (Pneumocystis DNA); lane 14, a negative control (water). (A) Oral swabs collected before rats were immunosuppressed. (B) Oral swabs collected at rat death. (C) Lung homogenate samples for each rat.

FIG. 3

PCR analysis showing Rcc primer sensitivity for P. carinii f. sp. carinii plasmid template. Lanes 1 to 11, P. carinii f. sp. carinii plasmid concentrations ranging from 1 to 10⁻¹⁰ ng (theoretical one plasmid). Lane 12 is the positive control (Pneumocystis DNA). Lane 13 is the negative control (water).