Reducing the use of laboratory animals has been a long-term goal in biological research. Many in vivo assays, like rabbit endotoxin testing or mouse antibody production testing to detect viral contaminants have largely been replaced by in vitro enzyme or PCR-based assays. And although some in vitro assays have proven inadequate replacements for a living, breathing whole animal system, a common rodent-dependent in vivo system that relies on hundreds of thousands of rodents annually is now in rapid decline.

This tectonic shift didn’t occur overnight.

Rather than having to lose valuable rodents used in their studies, researchers have long depended on soiled bedding sentinel rodents to indirectly monitor for infectious agents that can confound studies.

Here’s the concept. Commonly excluded viruses, bacteria and parasites are transmitted through weekly or bi-weekly transfers of soiled bedding from study mouse cages to sentinel mouse cages containing specific pathogen-free rodents. After three months, classical diagnostic methods including serology, bacterial culture and parasitology techniques are used to screen sentinel rodents for infectious agents.

This system has been the gold standard for monitoring laboratory rodent colonies for over 75 years, and for good reason. Before 1990, researchers housed rodents in open-top cages that allowed infectious agents to move relatively freely in dust and fomites. Biosecurity techniques were inadequate and untreated husbandry materials, such as bedding and feed, standard practice.

It’s different today. Biosecurity is better. Open-top caging is less common than individually ventilated microisolator caging (IVC), and the use of decontaminated cage changing stations mitigate the free movement of agents from cage to cage. Autoclaves, irradiation and other methods sterilize surfaces, rooms, cages and husbandry supplies, minimizing infection or re-infection.

Through all this change, sentinel systems also faced several new challenges. The sharing of transgenic mouse models among facilities led to shared rodent infectious agents arriving with the mice. Furthermore, while IVC caging restricts the spread of agents, it has become more evident that many agents do not transmit efficiently through soiled bedding, and as a result go undetected.

And if bedding sentinel mice couldn’t efficiently detect excluded agents in a quarantine situation, what might that mean for their ability to monitor research colonies when only a fraction of soiled bedding from each study cage was transferred? Failed agent detection in bedding sentinels is no more evident than for fur mites, which can cause mice to self-mutilate through scratching. Because the parasites do not efficiently transfer to bedding sentinels, they can go unnoticed in rodent colonies for years.

The need to improve agent detection and the evolution of affordable high-throughput PCR technology eventually persuaded laboratories to accept and steadily replace bedding sentinels with PCR testing of pooled non-invasive samples obtained directly from quarantined mice (oral swab, body swab and fecal pellet). This eliminated the need for bedding sentinels, improved detection of agents, reduced the total quarantine time from three months to two weeks, enabled research to start sooner and quickly allowed isolation of quarantined mice infected with excluded agents. Some receiving vivariums now require mice be tested before they are shipped to prevent arrival of unwanted agents.

Dusting for pathogens
PCR-based pathogen surveillance of vivariums can now be taken to an even higher level using exhaust air dust (EAD) testing. Dust samples can be collected from compatible IVC racks and evaluated using PCR panels potentially eliminating the need for soiled bedding sentinels entirely.

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

Consider those elusive fur mites. In 2013, Dr. Eric Jensen at the Medical College of Wisconsin demonstrated that EAD collected from an IVC containing a single cage of low-infestation mice could be consistently detected by PCR within two to eight weeks.

Our laboratory has been independently and collaboratively investigating EAD to determine the advantages and challenges. On the plus side, EAD PCR testing eliminates the need for sentinel mice, which can be a significant cost savings. Most importantly, it detects nucleic acid for agents that infect sentinel mice, and many more agents that don’t.

Fine-tuning exhaust air dust testing
It’s important to note that not all IVC racks are equal. Racks that have uninterrupted airflow from cage to dust collection points—often exhaust hoses and plenums—tend to be the best racks to use. For these racks, our investigations and field data support that EAD PCR testing detects all agents in the bedding sentinels and many others, too.

Other IVC racks have filters disrupting the free-flow of dust particles into exhaust plenums, which means materials containing infectious agent nucleic acid may not migrate or may have a delayed migration out of the cages to dust sampling or collection sites. A dust collection filter, first described by Susan Compton at Yale in 2004, may concentrate the dust to improve IVC racks with cage-level filtration. We found that on one IVC rack model with cage-level filtration, a filter placed in front of the exhaust HEPA pre-filter was still able to detect more agents than found in the bedding sentinel by traditional methods or PCR.

In racks with a filter-obstructed airflow at the cage-level, it’s necessary to test the sentinel cage filter the air flows through. Although, this doesn’t eliminate bedding sentinel cages, it does eliminate the need to ship sentinels, resulting in considerable cost and time savings. Our studies also demonstrated that the fecal material transferred to the sentinel cages brought along adequate agent nucleic acid that could be detected on the sentinel cage filter even though the sentinels never became infected with a large number of agents detected on the filter.

When using EAD PCR it’s important to remove residual nucleic acid during routine cage and rack cleaning because it can lead to a false-positive result in subsequent testing. EAD PCR is also very sensitive, which allows it to pick up low copy infections, but also makes it prone to false-positive results. In the event of unexpected results (positive or negative finding) the testing laboratory should be contacted to discuss confirmatory testing.

While challenges remain, PCR-based strategies for direct testing of quarantined rodents and for monitoring dust can replace, reduce or refine the use of sentinel mice in the infectious screening process, supporting the 3R philosophy while greatly improving the detection of rodent infectious agents to help keep animal colonies safe.