Thanks to advances in PCR, rodent sentinels may not be necessary.

Rodents are ubiquitous in research. They are also precious to science so laboratories invest a lot of time and money making sure that these research colonies, enlisted to find cures for diseases, aren’t getting exposed to unwanted pathogens that can compromise their health and the health of the study.

Traditionally, surveillance has involved putting test mice or rats in contact with research animals’ bedding material. Once these control animals, referred to as “sentinels,” have had enough time to potentially produce an immune response to any pathogen (usually 12 weeks), they are sent to a rodent diagnostic lab.

As an alternative method, laboratories also use PCR—which dawned about 30 years ago and is now commonplace in most animal diagnostic units. For years, labs relied on PCR methods that used gels to separate and amplify the DNA and RNA of suspected pathogens. But the advent of fluorogenic PCR 15 years ago, fed the way for the development of new advanced platforms. One of these platforms, the OpenArray® (Life Technologies), for which Charles River played the role of an early adopter, burst onto the scene 10 years ago and has since made the process of sentinel bedding or even first-generation PCR feel like the proverbial Model T. The fluorescent PCR technology used by CRL is called TaqMan, whose name was derived from the videogame, PacMan, because of the way the polymerase was portrayed to “consume” a hybridization probe. In a nutshell, Taq Polymerase, which is found in bacteria that live in very hot conditions, cleaves dual-labeled probes that help to improve the specificity of quantitative PCR. The PCR platforms consist of a chip the size of a microscope slide that contains over 2,500 individual assays, and is the engine behind PCR rodent infectious agent (PRIA®) panels. When filled with an astonishingly small 33 nanoliter reaction cocktail, each assay can detect as few as 1-10 copies of bacteria, viruses or parasite nucleic acid per assay isolated from the submitted sample types (see Feb. 27, 2012 Eureka blog).

Because the PRIA® panels are used to directly test the animals in question for quarantine situations, sentinels are not required, which reduces the amount of animals needed. But an even hotter trend in rodent vivarium diagnostics—PCR-based pathogen surveillance of entire spaces—is taking environmental monitoring to an even higher level by administering the biological equivalent of the white-glove test. Referred to as exhaust air dust (EAD) testing, dust samples can be collected from compatible IVC racks and evaluated using PRIA® panels potentially eliminating the need for soiled bedding sentinels.

TaqMan technology uses swabs of dust accumulation from air exhaust to monitor 100 or more cages at a time with much greater sensitivity than using a cage of “sentinel” mice housed on bedding samples from each test population. This improves test sensitivity, decreases the time from infection to detection, and does away with the need for “sentinel” mice, thus increasing the number of cages available to further the research mission.

A handful of animal diagnostic labs have been monitoring exhaust ducts and other surfaces since the 1990, and PCR analysis of gauze filters placed in the exhaust system has been used to detect mouse hepatitis virus and Sendai virus. But it’s the more recent refinements in PCR technology that make this surveillance tool a potential game changer, particularly for low-prevalence pathogens, or organisms that are not usually transmitted by rodents via dirty bedding. For instance, a recent study1 led by Eric Jensen of the Medical College of Wisconsin’s Biomedical Resource Center took advantage of a new, more sensitive PCR assay specific for murine fur mites to look at whether collecting air exhaust samples from an individual ventilated caging (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 almost 95% of the time over a four-week period.

And an internal study conducted recently at Charles River and presented last fall at the Federation for Laboratory Animal Science conference, used a PCR rodent infectious agent (PRIA®) panel of commonly reported or excluded pathogens to sample exhaust air. The results detected 13 agents, including several that were previously shown not to transmit efficiently to bedding sentinels and were not detected by previous sentinel use, such as the Jawetz and Heyl strains of P. pneumotropica and Cryptosporidium.

Like any strategy, sampling air exhaust and other environmental monitoring has its challenges. Not all cage racks are created equal; some IVC racks don’t collect much dust and so you need to know where the dust gathers to test properly. Swabbing static microisolation cages or cages where exhaust air is filtered before it is sampled won’t be effective.

And more data are needed comparing different testing methods. A 12-month blinded study launched last year at seven leading academic animal facilities is also comparing air duct samples in cage racks with sentinel bedding, and depending upon the results it could dramatically reduce the need for sentinel animals in routine surveillance.

And perhaps go a long way in helping laboratories adhere to the 3Rs.

If you are interested in hearing more about environmental monitoring in animal research facilities, you might find this webinar series by Charles River scientists helpful.

References:

  1. J. Am Assoc Lab Anim Sci. 52(1) 28 2013