Detection of Pneumocystis carinii in rat colonies by PCR and Serology

1. Detection of Pneumocystis carinii in rat colonies by PCR and Serology • Vandana S. Dole, Michelle L. Wunderlich, Barry A. Bronson, Robert L. Brouillette, Rajeev K. Dhawan, William R. Shek, Kenneth S. Henderson*

2. Background • Elwell, et. al. 1997 described distinct lung lesions characterized by lymphohistiocytic interstitial pneumonia and perivascular lymphocytic cuffing in laboratory rats • Albers et. al. (Vet. Path, 2009), published evidence supporting it was caused by a transmittable agent and suggested diagnostic histological criteria for infectious interstitial pneumonia • Pneumocystis carinii identified as the etiologic agent for the lesions (Livingston et. al., Comp. Med. 2011) and that it was not a virus as was previously reported • Our laboratory corroborated the identity of the etiology with the intent to improve methods for screening through a series of investigations (NAALAS, 2010).

3. Identification of the etiology was required so that more efficient, sensitive, and specific methods of screening could be developed • For more than a decade, only histopathology was available for screening • Labor intensive • Small window of opportunity for detection of lesions, which begin to resolve 5-6 weeks after the initial peak • Albers et. al. study • Naïve rats introduced into infected breeding colony • Exposed to 50% soiled bedding from rats  8 weeks of age

4. Overlay of histology scores with quantitative PCR, and IFA serum titration data obtained for archived samples • Genome copy numbers (PCR) increases slowly and the peak coincides with lung lesions • Henderson, et. al., 2010, NAALAS Seroconversion occurs when PCR and lesion peak Copy numbers decrease and lesions begin to resolve shortly after seroconversion

5. Detection challenges for P. carinii • Pneumocystis transmission is not efficient • Pneumocystis researchers commonly rely on intra-tracheal inoculation for consistent infection even for immunodeficient or immunosuppressed models • Rate of infection is related to dose • Seroconversion by exposure to bedding or open top cage environments is delayed compared to contact exposure (Henderson, et. al., submitted for publication) • 4.5 day doubling time (Aliouate, et. al., 1999) • Seroconversion occurs ~ 7 weeks after rats received 50% dirty bedding in an open-top cage barrier room (Henderson, et. al., NAALAS, 2010)

6. Challenge for PCR Henderson, et. al., submitted for publication • CD rats exposed by contact to 2- week pre-exposed RNU rats • Different sample types evaluated by PCR • Lung is the best PCR sample • Provides the earliest and largest window of detection • Oral swabs (ante-mortem) are not suitable • Same for nasal swabs and fecal pellets (data not shown)

7. Challenges for Serology Seropositive Seronegative • Indirect immunofluorescence assay (IFA) • Requires propagation of the organism • Can not be continuously passaged in culture • Requires propagation in rats for antigen • Too labor intensive for primary screen • P. carinii MFIA development • Recombinant antigen (Dhawan, et. al., NAALAS, 2011) • Provides a high-throughput multiplexed screening of samples

8. Endemically infected barrier rooms Pathology/MFIA Scores • Henderson, et. al., 2010, NAALAS • Sprague Dawley Rats (N=6/age group) • MFIA detects low maternal antibodies in 3 wk rats • Antibodies mostly absent in 7-8 wk rats, but 80% are PCR + • All 10-12 wk rats were PCR positive and seroconversion is detected in 50% of rats • At 29-30 weeks all rats are MFIA positive.

9. 10 week time course study: CD rats exposed by contact Pathology/MFIA Scores • Henderson, et. al., 2010, NAALAS Similar pattern as observed for Albers et. al. study Time line was ~2-week earlier

10. Summary of test results for rats from endemically infected barrier room and the contact time-course study • Suggests that serology or PCR alone may not provide comprehensive detection a -Data are from tests of 132 contact-exposed CD rats in transmission experiments II and IV and of 36 SD rats in the enzootically infected barrier-room breeding colony (Fig. 9). b -Positive cutoffs: ≥ 5 Pneumocystis DNA copies/mg lung by qPCR and antibody titer ≥ 0.5 Log10 by P. carinii IFA. c - Different Greek letter superscripts denote significant differences (P <0.01, Cochran test with pair-wise comparisons and the McNemar’s test)

11. Question: Could bedding sentinel infection be delayed and seroconversion not detected by serology if the infectious dose is too low? • Based on our concerns, rats submitted to Research Animal Diagnostic Services (RADS) for routine health monitoring since January 2011 were monitored by both MFIA and PCR for P. carinii • Data from the RADS Internet Laboratory Information Management System (ILIMS) was extracted to determine prevalence and to compare PCR and MFIA screening methods

12. Independent assay screening: Samples submitted for PCR or MFIA External samples received by RADS 1/2011 – 5/2011

13. Parallel screening of rats by PCR and MFIA n=599, distribution of 131 positives Pneumocystis PCR and P. carinii MFIA results were compared for rats submitted to RADS for routine health monitoring Combined Prevalence = 20%: Compare to 6% reported based on lung screening by histology (Pritchett-Corning, et. al., 2009)

14. Examples of rat submissions • Detection by one method or the other is not uncommon within sponsor submissions • Other variables that may contribute to delayed seroconversion in bedding sentinels • Prevalence on the rack • Stage of infection in study rats • Amount of bedding that is transferred

15. Summary P. carinii is a highly prevalent infectious agent in laboratory rats P. carinii replicates slowly and is best spread by direct contact Our previous studies suggest that that the timeline for sero-conversion is dose dependent Serology and PCR together are important for detection of P. carinii