Sanitation for Environmental Health Monitoring

When using Environmental Health Monitoring (EHM) techniques such as exhaust dust testing (EDT) or sentinel-free soiled bedding (SFSB), adequate sanitation of IVC racks and cages is essential to avoid legacy nucleic acids on surfaces that can cause false positive results. However, for routine testing of successfully excluded rodent pathogens and known agents within rodent colonies, the nuance of IVC rack and cage sanitation are likely inconsequential.

When this becomes important is when an undesirable agent is introduced, or efforts to exclude an existing agent from a subpopulation of rodents within a facility is desired. It is as this point when the stability of legacy nucleic acids from agents that are no longer present confound the use of PCR diagnostics to detect the reintroduction or continued existence of an agent.1 

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.4 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.3 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.8 

For agents which shed copious amounts of genetic material into a racks exhaust system, 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.5 If IVC rack autoclaving is possible, cage wash practices followed by autoclaving has also been demonstrated to be effective for Rodentibacter spp.6 and Helicobacter hepaticus.7 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.9,10

Data for the assessment of legacy nucleic acids within rodent cages is 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. A recent publication has illustrating the effectiveness of standard cage wash practices for removal of nucleic acids for the rodent pathogen MKPV.2 However, additional data is needed for the evaluation of sanitation practices for cages that ultimately will be used to hold soiled bedding for SFSB applications.    

References

  1. Bauer, B. A., Besch-Williford, C., Livingston, R. S., Crim, M. J., Riley, L. K., & Myles, M. H. (2016). Influence of Rack Design and Disease Prevalence on Detection of Rodent Pathogens in Exhaust Debris Samples from Individually Ventilated Caging Systems. Journal of the American Association for Laboratory Animal Science: JAALAS, 55(6), 782–788.

 

  1. Carlson, A. L., Floyd, R. J., Ricart Arbona, R. J., Henderson, K. S., Perkins, C., & Lipman, N. S. (2022). Assessing Elimination of Mouse Kidney Parvovirus from Cages by Mechanical Washing. Journal of the American Association for Laboratory Animal Science: JAALAS, 61(1), 61–66. https://doi.org/10.30802/AALAS-JAALAS-21-000096

 

  1. Clancy, B. M., Theriault, B. R., Schoenberger, J. M., Bowers, C. J., Mitchell, C. M., Langan, G. P., Ostdiek, A. M., & Luchins, K. R. (2022). Identification and Control of an Ornithonyssus Bacoti Infestation in a Rodent Vivarium by Using Molecular Diagnostic Techniques. Comparative medicine, 72(2), 113–121. https://doi.org/10.30802/AALAS-CM-21-000105

 

  1. Mailhiot, D., Ostdiek, A. M., Luchins, K. R., Bowers, C. J., Theriault, B. R., & Langan, G. P. (2020). Comparing Mouse Health Monitoring Between Soiled-bedding Sentinel and Exhaust Air Dust Surveillance Programs. Journal of the American Association for Laboratory Animal Science: JAALAS, 59(1), 58–66. https://doi.org/10.30802/AALAS-JAALAS-19-000061

 

  1. Manuel, C. A., Pugazhenthi, U., & Leszczynski, J. K. (2016). Surveillance of a Ventilated Rack System for Corynebacterium bovis by Sampling Exhaust-Air Manifolds. Journal of the American Association for Laboratory Animal Science: JAALAS, 55(1), 58–65.

 

  1. Miller, M., Ritter, B., Zorn, J., & Brielmeier, M. (2016). Exhaust Air Dust Monitoring is Superior to Soiled Bedding Sentinels for the Detection of Pasteurella pneumotropica in Individually Ventilated Cage Systems. Journal of the American Association for Laboratory Animal Science: JAALAS, 55(6), 775–781.

 

  1. Miller M, Ritter B, Zorn J, Brielmeier M. 2016. Exhaust Air Particle PCR Detects Helicobacter hepaticus Infections at Low Prevalence. Journal of Veterinary Science & Technology 7. https://doi.org/10.4172/2157-7579.1000343
  2. Pettan-Brewer, C., Trost, R. J., Maggio-Price, L., Seamons, A., & Dowling, S. C. (2020). Adoption of Exhaust Air Dust Testing in SPF Rodent Facilities. Journal of the American Association for Laboratory Animal Science: JAALAS, 59(2), 156–162. https://doi.org/10.30802/AALAS-JAALAS-19-000079

 

  1. Ragland, N. H., Miedel, E. L., Gomez, J. M., & Engelman, R. W. (2017). Staphylococcus xylosus PCR-validated Decontamination of Murine Individually Ventilated Cage Racks and Air Handling Units by Using ‘Active-Closed’ Exposure to Vaporized Hydrogen Peroxide. Journal of the American Association for Laboratory Animal Science: JAALAS, 56(6), 742–751.

 

  1. Ragland, N. H., Miedel, E. L., & Engelman, R. W. (2019). PCR Prevalence of Murine Opportunistic Microbes and their Mitigation by Using Vaporized Hydrogen Peroxide. Journal of the American Association for Laboratory Animal Science: JAALAS, 58(2), 208–215. https://doi.org/10.30802/AALAS-JAALAS-18-000112

FAQ About Sanitation

Should I worry about legacy nucleic acids after washing IVC racks or cages?

Yes. PCR assays are very sensitive, and can detect old, or legacy nucleic acids of agents that may no longer be present. This can make interpreting results difficult. Data suggests that you can take a stepwise approach to removing legacy nucleic acids if it is causing problems. This can include manual scrubbing in addition to standard sanitation practices, washing equipment 2 or 3 times in a mechanical washer, use of bulk autoclaves for cage and rack sterilization, and even vaporized hydrogen peroxide (VHP) exposure to degrade legacy nucleic acids on equipment.