PCR testing is so sensitive that it can detect old, legacy nucleic acids of pathogens that may no longer be present. When these are present, you may need to employ special attention to sanitation practices. Additional practices can include manual scrubbing, multiple washes, autoclaves, and even vaporized hydrogen peroxide to adequately degrade legacy nucleic acid.
Adequate sanitation of IVC racks and cages is essential when using Environmental Health Monitoring (EHM) techniques such as exhaust dust testing (EDT) or sentinel-free soiled bedding (SFSB). Sanitation is necessary to avoid the possibility of false positives due to legacy nucleic acids on surfaces that can be present even after an agent has been excluded from a population of rodents.
Sanitation is less critical when performing routine testing of successfully excluded rodent pathogens and pests within rodent colonies. In these cases, there is unlikely to be any legacy nucleic acid present due to successful exclusion. Therefore the nuances of IVC rack and cage sanitation are likely inconsequential.
Conversely, attention to sanitation practices becomes critical when an undesirable agent is introduced or when there are efforts to exclude an existing agent from a subpopulation of rodents. In these cases, legacy nucleic acids from agents that are no longer active can confound the use of PCR diagnostics to detect the reintroduction or continued existence of an agent.
There have been a few publications regarding the sanitization of racks for use with EDT. Recommended methods depend on the particular pathogens of concern and known shedding levels. Mailhiot et al. showed that typical cage wash practices with temperatures of 180°F with detergents were adequate to remove nucleic acids for Helicobacter spp., Rodentibacter spp., and MNV from IVC racks and ensure negative rack plenum swab PCR results.3 At the same institution, manual scrubbing of the plenums with a detergent before cage wash was used to remove Ornithonyssus bacoti adhered eggs to eliminate legacy nucleic acids.2 In addition, Pettan-Brewer et al. described the use of dedicated IVC racks for higher barrier rooms or the use of two rounds of cage wash practices to prevent legacy nucleic acids for Helicobacter spp. and MNV.7
For agents which shed copious amounts of genetic material into rack exhaust systems, such as Corynebacterium bovis, Manuel et al. found that a single cycle through a 180°F rack washer, using detergent, was not adequate to remove legacy nucleic acids. However, autoclaving the racks demonstrated complete success as determined by C. bovis specific PCR.4 If IVC rack autoclaving is possible, cage wash practices followed by autoclaving have also been demonstrated to be effective for Rodentibacter spp.5 and Helicobacter hepaticus.6 If whole rack autoclaving is not available, vaporized hydrogen peroxide sanitation of IVC racks and rack blowers has been shown to eliminate microbial nucleic acids so that none are detected by PCR.8,9
Data for the assessment of legacy nucleic acids within rodent cages are not as abundant as previously described for IVC racks. Yet, testing of pooled soiled bedding within a sanitized cage by PCR also requires that the cage be free of legacy nucleic acids. Pre-irradiated and -bedded disposable caging, in addition to vendor-prepared dedicated collection boxes, are available to potentially alleviate these concerns but may not be desirable for use in every institution. A recent publication illustrated the effectiveness of standard cage wash practices for the removal of nucleic acids for the rodent pathogen MKPV.1 However, additional data is needed for the evaluation of sanitation practices for cages or containers that ultimately will be used to hold soiled bedding for SFSB applications.