PCR-based Pathogen Screening of Lab Rodent Vivaria Works

With screening methods available for improving infectious agent detection in vivariums, and a push to reduce animals in research, it’s time we stopped using soiled bedding sentinels

A substantial and convincing volume of data is now available showing that PCR-based sampling schemes for both quarantine and routine health monitoring of rodents improve infectious agent detection better than traditional use of soiled bedding sentinels. The transfer of soiled bedding from colony research mice to a cage with clean mice, and the hopes that infectious agents transfer and subsequent diagnostic detection, has been the gold standard for decades.

As the use of soiled bedding sentinels has been identified as a flawed system, newer PCR-based methods have been investigated and have shown that most improve infectious agent detection with the additional advantage of reducing or eliminating the need for live rodents as part of the sampling process. Yet a large percentage of the lab animal community still struggles with the change to non-sentinel methods despite the advantages and accessibility.

The concern with soiled bedding sentinels was first apparent from outcomes in a study my colleagues and I conducted at Charles River, which compared direct sampling of mice for PCR to traditional soiled bedding sentinel in an emulated quarantine. The large scale of agents that went undetected in sentinel mice prompted the beginning of our recommending PCR-based quarantines. Today, the majority of mice that are shipped to research vivaria from other institutions for quarantine are directly sampled for PCR testing. 

From Exhaust Air Dust PCR to Collection Media

Among the first PCR samples investigated for routine health monitoring was exhaust air dust (EAD^®) collected downstream from the exhaust plenums of individually ventilated cage (IVC) racks.  A pool of swabs at a few specific locations could effectively monitor all the cages on the rack.  Two IVC rack manufacturers took it a step further by creating in-line filter holders that simplified the EAD collection process.

Unfortunately, EAD PCR testing is only accommodated by (IVC) racks without exhaust filters at the cage level, so the majority of IVC racks and static microisolator caging used for research mice were unable to effectively use EAD PCR. Methods of testing the soiled bedding sentinel cage-level filter were developed that also improved agent detection over soiled bedding sentinels, but these methods were cumbersome and had limited adoption. 

More recently, collection media, commonly a filter material or swab, have been used to bind infectious agent-associated dust particles directly from pooled soiled bedding for PCR testing.

The methods require the collection of a small amount of bedding as is done for soiled bedding sentinels, which is then agitated with or swabbed by the collection media. As with EAD, this method demonstrated superior detection of infectious agents over the use of soiled bedding sentinels and eliminates the need for live animal in the sampling process. This method for PCR sample collection is also universal so it can be used regardless of housing system. 

PathogenBinder^® Enters the Picture

Charles River has standardized the sample collection process with PathogenBinder^®, which uses a protocol and contact media that optimizes infectious agent detection and reduces variation in the sample collection process as well as the diagnostic laboratory’s processing of the sample. The PathogenBinder^® study used to optimize the process showed that the optimal conditions detected nearly three times the number of infectious agents versus soiled bedding sentinels. At the time this document was published, over 220 institutions have trialed or have implemented PathogenBinder^®. 

PCR-based method can now be applied to any cage type, has been reported to cost the same or less to operate, and greatly improves detection of prevalent and commonly excluded agents. It is also an ethically responsible method for replacing a flawed animal-based procedure.

So, what are animal research facilities waiting for?

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