Detection and Remediation of Pneumocystis murina Infections by Environmental Health Monitoring

Animals and environment.

Research mice are housed at 3 primary facilities on the University of Colorado Anschutz Medical Campus, which is fully accredited by AAALAC International (no. 00235) and PHS assured (no. D16-00171). Only facilities I and II, containing ∼11,000 and 10,000 mouse cages, respectively, are involved in this case report. Mice in facilities I and II are housed in JAG 75 cages on individually ventilated MicroVent racks (Allentown LLC, Allentown, NJ) with ∼40 air changes per hour. The MicroVent racks are designed to supply air to each cage and exhaust a combination of cage and room air from near the cage lids. Racks are sanitized and replaced on a 6-mo cycle, which includes opening and spraying out plenums, mechanical wash (Lynx, Spire Integrated Solutions, Orlando, FL), and autoclaving (AMSCO series, Steris, Dublin, Ireland). All mouse cages in these facilities are autoclaved with aspen-chip bedding (Teklad Aspen Sani-Chips, Inotiv, West Lafayette, IN), a compressed cotton square (Ancare, Bellmore, NY), and a box or wire bar feeder. Mice are fed irradiated rodent Teklad diet 2920X (Inotiv). Hyperchlorinated reverse-osmosis drinking water is provided via an automated watering system (Avidity, Waterford, WI). Additional enrichment items, such as Bed-r-Nest, Mouse Igloo, and cardboard tubes, are autoclaved and made available and added to cages in housing rooms. Environmental controls include a set temperature of 22.0 ± 1 °C (72 °F), 30% to 40% humidity, at least 10 fresh-air changes per hour, and a 14-h light/10-h dark cycle. Mouse cages are changed every 2 wk. All husbandry procedures are performed in a HEPA-filtered workbench (ATS or ATS2, Allentown) within animal housing rooms. A disposable hair bonnet and isolation gown or dedicated scrubs are required to enter the animal facilities, and nitrile gloves are worn when opening cages. Clidox-S (Pharmacal, Naugatuck, CT) mixed 1:18:1 or Peroxigard (Virox, Oakville, ON, Canada) are used to disinfect all work surfaces and gloved hands while working with the animals. Research mice are purpose-bred inhouse, sourced from commercial research animal breeders, or imported from other research institutions following a quarantine period. All mouse populations discussed in this report are under IACUC-approved research protocols at the University of Colorado Anschutz Medical Campus.

Sentinel-free health monitoring program.

MicroVent racks are fitted with a Sentinel 1 (Allentown LLC, Allentown, NJ) media holder that is sterilized as part of the rack every 6 mo (see Animals and environment). Facility I has 59 double-sided mouse racks and 151 single-sided racks. Facility II has 25 double-sided and 169 single-sided racks. Quarterly, Sentinel media (Allentown) from mouse racks are tested for mouse pathogens by PCR (Table 1), including a comprehensive annual panel, which includes P. murina (IDEXX BioAnalytics, Columbia, MO).

Confirmatory testing.

When a rack tested positive for P. murina, confirmatory testing was performed at the rack level by single-agent PCR (IDEXX BioAnalytics) on Sentinel media in place for ∼1 mo. Racks that were confirmed positive were then tested using serial PCR sampling designed to determine the location of the infected cages on the rack without testing all cages. First, swabs were collected at the row level using a single flocked swab (PurFlock Ultra with breakpoint, Puritan Medical Products, Guilford, ME) in either the horizontal exhaust manifold (HEM) or along the exhaust air ports located behind each cage along each row of the rack. While swabbing the HEM ports is technically less challenging, routine Corynebacterium bovis surveillance swabbing of a rack’s HEMs with a single swab could move DNA and contaminate subsequent rows and thus complicate interpretation of P. murina results.^37,38 Furthermore, both sides of a double-sided MicroVent rack exhaust into a common HEM, which prevents localization of positive results to a single side of a double-sided rack. In these situations, the exhaust air ports for each cage on a row were sampled using a common swab to represent row level testing. Immediately after the swab was taken, swabs were enclosed in a sterile 2-mL centrifuge tube (Safe-Lock, Eppendorf, Enfield, CT) and submitted for P. murina PCR testing (IDEXX BioAnalytics).

Cages from each P. murina–positive row were tested individually by PCR as previously described.³⁷ Briefly, the inside of the cage lid, wire bar feeder (if present), and walls of the back one-third of the cage were swabbed using a flocked swab and submitted for P. murina PCR in a centrifuge tube as described above. To minimize the risk of cross-contamination between cages in the ATS, the working surface was kept wet with disinfectant, and only one cage, one swab, and one centrifuge tube were open at a time.

In addition, confirmatory serology testing was performed on a single immunocompetent mouse from a subset of P. murina–positive cages. Using manual restraint and a 4- or 5-mm lancet (Goldenrod, MEDIpoint, Mineola, NY), a single drop of blood was collected on a dried blood spot sampling strip (Opti-Spot, IDEXX BioAnalytics) via puncture of the submental or facial vein and submitted for P. murina serology (IDEXX BioAnalytics).

Moving and retesting suspected false-positive colonies.

Low-to-medium relative copy numbers on racks 5 to 8 in case 3 were suspected to be false positives due to environmental contamination from the other high copy number racks in the room (Table 2). A 2-person method was used to change cages and relocate research laboratory J’s mouse colony of ∼200 cages to a new housing room. Cage changing was conducted in an ATS with one person designated ‘dirty’ (handled all caging from the P. murina–positive room) and the other ‘clean’ (handled all clean caging and mice). Mice were moved from their old cage into a new, autoclaved cage and placed directly on a clean rack outside the housing room, which was then moved into a previously empty mouse housing room. Each rack contained an Allentown Sentinel 1 collar (Allentown LLC, Allentown, NJ) and medium that was changed and tested monthly for P. murina for 3 mo until the colony was considered P. murina–negative and normal quarterly health surveillance resumed.

Necropsy and postmortem diagnostics.

Mice were euthanized via CO₂ asphyxiation and secondary bilateral thoracotomy consistent with the AVMA guidelines for the euthanasia of animals (2020 edition). The airways were infused with 0.9% normal saline via the trachea to expand the airways without affecting DNA integrity. The left lung was fixed in 10% neutral buffered formalin and prepared for histology. Slides were hematoxylin and eosin and silver stained to assess morphology and identify Pneumocystis cysts (IDEXX BioAnalytics). The right lung lobes were frozen in a centrifuge tube and tested via PCR for P. murina (IDEXX BioAnalytics).

Case 1.

In facility I, 3 racks tested positive for P. murina on the first annual panel of EDT media that had been in place for 3 mo (Figure 1A). Two of the racks had >100 copies of P. murina DNA detected while the other rack had only 2 copies detected, suggesting a possible false-positive in the third rack. Replacement media that had been in service for 1 mo was submitted for single-organism PCR testing and confirmed that 2 of the 3 racks were positive (Table 2). These 2 double-sided racks housed mice from 6 investigators, 1 of whom had animals on both racks. To narrow down the source of P. murina DNA, rows were tested by swabbing the HEM as described above. Four rows tested positive across the 2 racks, with all housing mice from a single research laboratory (Table 3). A total of 11 different strains of mice were represented in these 4 rows, including several strains with genetic modifications affecting the immune system.

At this time, research laboratory D was notified of our findings, and we agreed to perform cage-level testing while the laboratory minimized cage movement. The racks included part of this laboratory’s breeding colony with all cage slots filled, so minimizing cage movement was not strictly enforced. Instead, cages from the positive-testing rows were labeled with a cage card sticker so they could be followed over time. Offspring of the breeding cages on positive rows were also labeled for additional testing, even if their new row had not tested positive by the initial swab. Blood was collected for serology and each cage was swabbed for PCR testing as described above.

Ten of 34 individually tested cages from laboratory D’s colony tested positive for P. murina. Eight were positive by PCR and 2 cages of recently weaned mice (∼1 mo old at time of testing) were positive by serology but negative by PCR. The remaining PCR-positive cages were also positive by serology except for one cage that had low immunoglobulin levels (likely insufficient sample). All P. murina–positive cages housed mice of the same immune-vague strain, called OT-1 PD-1 KO (100% of the laboratory’s cages of this strain). All adjacent cages containing other strains tested negative for P. murina by both PCR and serology.

The P. murina–positive mouse strain had been imported to the institution from a collaborator several years prior and had since become available commercially from research mouse vendors. The research laboratory elected to euthanize the mice from this strain and purchase them from a research vendor’s SPF colony for future studies.

Postmortem tissue samples were collected from mice from 3 separate cages, representing different ages (n = 3 mice/age) for lung PCR and histology as described above. The oldest group was 150 d old at euthanasia. Two of these 3 mice had gross lung changes consistent with P. murina, including a diffuse mottled gray-pink color and incomplete deflation after thoracotomy. All 3 were PCR positive for P. murina and had moderate-marked multifocal histiocytic alveolitis with foamy eosinophilic material and lesser multinucleated cells, lymphocytes, plasma cells, and granulocytes, consistent with P. murina infection. Another group of 3 mice was 108 d old at submission. Of these, one had gross lung changes consistent with P. murina. This mouse also had moderate-marked histology changes as described for the older mice. The other 2 had a mild-to-moderate versions of these changes, and all 3 tested positive for the agent by lung PCR. The third, youngest group of mice was just 36 d old at the time of euthanasia and necropsy for tissue collection. None of these mice had gross lung changes, but all 3 had mild-to-moderate histology changes consistent with P. murina, and all 3 tested positive for the agent by lung PCR.

Case 2.

In facility II, one room had a single rack that tested positive with 16 relative copies of P. murina DNA (Figure 1B). Retesting was performed as in case 1 via media exposed to exhaust air on the rack for 1 mo. This confirmed the positive test result with 31 relative copies (Table 2). On this rack, 7 of the 10 rows were occupied by mice from 2 different research groups. Row testing identified 2 positive rows from the same principal investigator, containing cages of wild-type C57BL/6 or Htr3a-GFP mice not expected to have any immune abnormalities. These 9 cages were tested by PCR swab and serology 12 d after the previous cage change to maximize potential for DNA accumulation in the cage. All of these cages were negative by serology. One cage tested positive by PCR, the offspring of a C57BL/6 × Htr3a-GFP breeding cage that tested negative by PCR. These offspring were ∼15 wk old at the time of testing. Other older and younger sibling cages were also tested and were negative by PCR and serology.

The research laboratory had completed the research project using these mice at the time of follow-up testing and elected to euthanize the remaining mice from the Htr3a-GFP line in the single-positive cage. The 2 breeding cages of this strain (6 and 9 mo of age at euthanasia) were submitted for histology and P. murina lung PCR. No lung changes were noted on necropsy and both pairs were negative for P. murina PCR and histology. The mice in the PCR-positive cage were not submitted for lung PCR or histology, but were presumed positive based on positive rack, row, and cage-level PCR test results.

Case 3.

In one housing room in facility II, all 7 racks tested positive on the first annual EDT PCR screening panel (Figure 1C). Three racks with all cages on the same research laboratory’s protocol had larger relative copy numbers on initial and follow-up testing compared with the other 4 racks, which housed cages on another investigator’s protocol (Table 2). As in cases 1 and 2, follow-up testing was performed on media in the racks for 1 mo following the initial positive test on 3-mo-exposed media.

Although all 7 racks tested positive both times, the high copy numbers in the first 3 racks and lower copy numbers in all subsequent racks suggested that test results from racks 5 to 8 may be false positives (Figure 1C). In discussion with the 2 research laboratories, we also learned that many of the strains on racks 1 to 3 had B cell abnormalities (altered immune system), whereas the strains on racks 5 to 8 were expected to be immunocompetent, and therefore less susceptible to P. murina infection.

The mice housed on racks 5 to 8 (suspected false positive) were moved to a different, previously empty housing room for isolation and testing. By removing these mice from the room with high levels of environmental contamination, we could more efficiently perform follow-up testing at the rack and/or row level with higher confidence in subsequent testing. The colony was moved by changing mice into new autoclaved caging and new autoclaved IVC racks using a 2-person method as described above. These racks were then monitored using media in the collar within the exhaust manifold, which was tested monthly for P. murina over 3 mo. All of these tests were negative, and the colony was released from isolation.

During this time, follow-up testing on racks 1 to 3 (Figure 1C) was performed by row swab testing followed by cage-level testing. All rack rows housing one or more cages of mice were tested, which included all 10 rows of all 3 racks for this colony. Rack 1 housed experimental cages, primarily offspring from breeders on racks 2 and 3. Rack 2 was ∼30% breeding cages and the rest were holding cages, and rack 3 was 90% pair breeding cages. Results of row testing are shown in Table 4.

Once the P. murina–positive rows were identified, cage-level swabbing was performed to identify affected cages by PCR. Because most of the strains had altered B cell biology, serology was not performed in case 3, as low serum immunoglobulin concentrations were anticipated and results would be difficult to interpret. As for case 1, the research laboratory in case 3 frequently moved cages on and between these 3 racks, so once a strain tested positive, other cages of this strain were considered suspected positive. Unlike case 1, the breeding lineage was not clear for all affected cages and strains.

Three of the laboratory’s 15 mouse strains (20%) were identified as P. murina negative after 2 rounds of cage-level testing performed 7 to 8 wk apart with all negative results (data not shown). These 46 cages were considered uninfected, and the mice were relocated to another housing room to prevent potential cross-contamination. These strains were also identified by the laboratory as having a normal immune system. Despite this, serology was not used, as more importance was placed on P. murina environmental shedding than serologic status. This rationale is supported by results from case 1 where the infected mouse strain was seropositive for P. murina but continued environmental shedding. Subsequent routine health surveillance of these 3 strains in the relocated housing room continued to produce negative P. murina results by EDT.

The remaining 12 strains had one or more cages that tested positive by cage-level PCR testing. These consisted of 6 strains that were considered by the laboratory to have a functional immune system (although they could identify subtle changes in immune cells on flow cytometry) and another 6 with known changes in B cell function. After testing positive initially, the latter group of 6 strains was assumed to be infected by P. murina and no additional testing was performed. The mice with more normal immune systems were retested 7 to 8 wk later. At that time, 1 of the 6 strains had been culled by the laboratory, as they were no longer needed for experiments. The remaining 5 strains all had multiple cages that retested positive by cage-level testing. Copy numbers ranged from 2 to 1,035 per cage (data not shown). Breeding mice from 3 of these strains were again tested at the cage level 7 mo after the initial cage testing and were again found to be positive (data not shown). This confirmed that these strains, although genetically considered immunocompetent, had not cleared the P. murina infection. As noted above, blood was not collected to characterize the antibody response of these mice to P. murina.

This colony remained isolated in place in the original housing room. The laboratory’s grant funding supporting these strains and line of research was completed and the colony was eliminated in January 2025. Until that time, to minimize environmental contamination and the potential of cross-contamination between housing rooms, the strains that continued to actively shed P. murina were placed on an antibiotic-supplemented diet containing both trimethoprim and sulfadiazine (TMS, Uniprim, TD.06596, Inotiv, West Lafayette, IN). Following the provision of the medicated diet, continued quarterly testing for P. murina by EDT no longer detected P. murina from media within previously positive racks after 6 mo of treatment. Continued quarterly testing for P. murina by PCR of IVC rack media did not identify any further spread of P. murina in the rooms functionally and physically adjacent to this room, and the negative-testing strains and colony removed from the room have continued to be P. murina negative by rack PCR since moving.