PCR of exhaust air dust is rapidly becoming more mainstream and might one day replace dirty bedding sentinels, a tremendous boost for the 3Rs.

A few years ago, veterinarians at Cornell University detected an unexpected organism lurking in one of its mouse barrier rooms. Hmmm… Mouse colonies maintained for biomedical research are not germ-free, but they are supposed to be free of specific pathogens that can harm the animal and bias the research process. The parasite pneumocystis, the pathogen in question, was one of those excluded pathogens, and it had escaped the attention of dirty bedding sentinels—mice that serve as biological sentries for the entire colony.

Pneumocystis can be devastating to research mice, particularly immunodeficient mice bred for immunology, cancer and infectious disease research. So staff at the Cornell Center for Animal Resources and Education (CARE), where the university’s animal colonies, including around 10,000 mouse cages, are maintained and cared for embarked on a small study to see if a new and still experimental tool that analyzes exhaust air dust from cages using polymerase chain reaction (PCR) could find the pneumocystis and whether it did it more efficiently than the standard method.

“It turns out the only way we could detect it reliably was on PCR of exhaust air dust or through direct PCR of lung tissue,” says Erin Daugherity, a clinical research veterinarian and assistant director of technical services at CARE. “Dirty bedding sentinel were never positive.”

These findings have hardly sunk the ship for dirty bedding sentinels. They are still a reliable surveillance tool for many other excluded pathogens. But Cornell, one of the largest research animal facilities in the US, now includes PCR on exhaust plenum dust as part of a hybrid system that also includes fecal PCR—sequencing organisms found in animal waste—and sentinels.  

They are not alone. Other large academic or commercial facilities are also using PCR of EAD routinely, and a few are even using it exclusively, says Ken Henderson, Senior Director of Laboratory Services at Charles River’s and a pioneer in the use of PCR of EAD.    

“I was always under the impression that about 20% of our users were using EAD. More recently, we are finding that around 30% to 40% are EAD samples only or EAD with some type of other component.”

Henderson thinks PCR of EAD is rapidly becoming mainstream. He also believes PCR of EAD, coupled with other alternative health monitoring methods, will one day replace sentinels, a tremendous advance from an animal welfare perspective.

HEPA filters, fur mites and the evolution of EAD

Although EAD testing by PCR has been reported for rodent pathogen detection since the mid-90s, it is only recently that large panels of PCR assays comprising the entire list of commonly excluded agents became available, opening the door to replacing traditional screening methods now used to monitor bedding sentinels.

Henderson recalls that about 20 years ago veterinarian William White, who at the time oversaw Charles River’s diagnostic and professional services activities as well as its biosecurity program, approached him about using PCR to analyze filters in the barrier rooms for rodent production areas. Henderson’s team did and discovered that they could track parvovirus around the facility on filters. They also learned, luckily, that HEPA filters were largely doing a great job of keeping agents out of their barrier rooms.

Which begged the question: How did the parvovirus, which is difficult to eradicate, find its way into the facility?  The laboratory facility began using dust to look at parvovirus on keyboards, ventilation fans, door knobs and other surfaces, and eventually demonstrated that the germ was moving through air, says Henderson.

Other groups had also become curious about EAD. A decade ago, Susan Compton, a research scientist with Yale University’s Department of Comparative Medicine, did PCR analysis on gauze filters placed inside the exhaust system of ventilated cages. It took research by Eric Jensen of the Medical College of Wisconsin’s Biomedical Resource Center to make the strategy practical. Jensen demonstrated that you could look for fur mites by monitoring EAD on the individually ventilated caging (IVC) racks that housed their research animals.

Jensen took advantage of a new, more sensitive PCR assay specific for murine fur mites to look at whether collecting air exhaust samples from an IVC system could be used to monitor murine fur mites rather than relying on sentinel mice. Fur mites are often poorly detected in bedding sentinels, but this study, which Charles River collaborated on, found that swabbing and testing shelf exhaust manifolds of IVC racks picked up the presence of murine fur mites over a four-week period almost 95% of the time.

“That immediately raised the question that if you can do it for one can you do it for others. And the answer was that you could,” says Henderson. “But for the laboratories that were looking at this and watching us it still seemed almost like science fiction at first at first.

Henderson found various academic and commercial collaborators willing to test the limits of PCR of EAD, and assisted many other facilities encountering challenges with sentinel testing. The findings from many of these pilot studies, which were informative and encouraging, helped to build credibility and push the technology past the pilot stage to routine monitoring.

There’s no magic bullet

Daugherity, who has been working with research rodents for close to 15 years, says the process of using dirty bedding sentinels works like this. A tiny scrap of bedding from every cage in the room gets transferred to designated sentinel cages. If the sentinel receives bedding containing a particular pathogen, it will hopefully mount a serological response or actually become infected, alerting the facility that the colony needs to be checked. In her facility, three dirty bedding sentinels are used for every 80 cages of mice.

The system is generally reliable, but Daugherity says it is not the most reliable method in every situation. Sometimes it misses bugs entirely. The fur mite case certainly proved that, but so did a separate incident of pinworms that also surfaced in one of Cornell’s barrier rooms. Like the fur mites, the pinworms had originated in a quarantine unit.

“I’m very proud of our quarantine system but there is no perfect quarantine program,” says Daugherity. “That is one of the ways that pathogens can come into your facility. We found that the [pinworms and fur mites] had been in our facility since 2014 and we never detected them using the dirty bedding sentinels.”

Even after they identified them using PCR of EAD, they still couldn’t find it on the dirty bedding, added Daugherity.

Yet PCR of EAD isn’t foolproof, either. The inconsistencies seem to occur with viral agents primarily, says Daugherity. Her group recently compared dirty bedding sentinels and PCR of EAD in norovirus detection, and found quicker results with the sentinels. “That’s a major concern because we want to make sure we monitor for our viruses,” says Daugherity. “That is why we can’t completely eliminate dirty bedding sentinels.”

Cage design also matters. PCR of EAD works best on IVC racks that have uninterrupted airflow from cage to dust collection points, such as an exhaust hose or plenum. IVC racks with filters disrupting the free-flow of dust particles into exhaust plenums can slow down or even prevent materials containing infectious agent nucleic acid from migrating out of the cages to dust sampling or collection sites.

“Because we have static microisolator or conventional cages at Charles River, we have developed ways to improve testing which are not EAD-supported but still allow us to find out what is on the rack,” says Henderson.

Residual DNA on the racks can also lead to false-positives, says Daugherity, so her facility has had amend the way they clean their racks.

Still, she feels quite confident about the results they are getting with PCR of EAD, and she’s excited about the impact it is having on reducing, replacing and refining the use of research animals, otherwise known as the 3Rs.

Her facility has been able to cut back on the number of sentinels by doing serology testing every other quarter rather than every quarter, and they no longer ship live bodies to diagnostic facilities, which means less stress on animals.

 “From an animal welfare perspective, it makes me incredibly happy and proud of our profession that we are trying to identify methods that detect pathogens in a better way and a more efficacious way,” says Daugherity. “It protects the animals, and allows the researchers to perform their research in a better way.”

How to cite:

McEnery, Regina. Robinson, Laura. The Dust Collectors. Eureka blog. Nov. 2, 2016. Available: http://eureka.criver.com/the-dust-collectors-video/