Photo from Pettan-Brewer et al. 2020. Adoption of Exhaust Air Dust Testing in SPF Rodent Facilities. JAALAS. 59(2), 156-162.

It is important to ensure that rodent colonies are free of adventitious infectious agents to support high quality, robust research & safety data. Over the last 50 years, health surveillance has been typically performed using sentinel animals. More recently, molecular-based diagnostics combined with environmental monitoring strategies have been adapted by many institutions to either supplement or replace the more traditional methods. These newer approaches have the possibility of significantly reducing/replacing the number of rodents used in maintaining specific-pathogen-free colonies.

What are some reasons to consider switching from soiled bedding sentinels?
  • 3Rs, replace thousands of mice/year
    • Improve compassion fatigue (i.e., “one well-being”)
  • Improve pathogen detection
    • Detection of excluded infectious agents. Some agents are not transferred well to sentinels via soiled bedding such as fur mites, Sendai virus, Lymphocytic choriomeningitis virus (LCMV), Lactate Dehydrogenase Virus (LDV), Rodentibacter pneumotropicus and R. heylii (previously Pasteurella pneumotropica),Helicobacter, etc.
    • Detect critical pathogens earlier (e.g., bovis, Manuel, 2017)
    • PCR may be more sensitive at detecting low level prevalence agents
    • Sentinel variability such as age, strain, and immune status can impact transmission and subsequent detection
  • Potential savings in money and/or labor
    • Avoid technical/staff inconsistencies innate in soiled bedding sentinel programs
  • Differential sampling can allow you to determine which specific cages are positive
  • Eliminate risk of receiving sentinels infected with excluded agents
How can environmental monitoring replace or be a compliment to soiled bedding sentinels?

Individually Ventilated Cages (IVCs) + Plenum Swabs

  • Advantages = Initially less costly than exhaust plenum media holders also referred to a “collars mounted media.”
  • Challenges = Some rack brands/designs are easier to implement environmental monitoring than others (e.g., not effective with rack designs with cage level exhaust air filtration). May not be sensitive enough for pathogens that are at low prevalence.

IVCs + Collar Mounted Media

  • Advantages = Operator variability is minimized & minimal handling is required. Detects Helicobacter & MNV with similar sensitivity to swabbing. Media helps increase dust collection. Acceptable for 3-month collection. Provides a history of pathogens that were present over the previous 3 months. Presumably reduced ergonomic effort.
  • Challenges = Can be physically difficult to place/collect collar mounted media from single-sided racks. Collars/media must be placed AFTER racks are washed & collected BEFORE wash which requires coordination (alternatively you can wait to wash racks until they test positive and then clean once potential outbreak is resolves). May be costly.

IVCs or Static Cages + Filter Paper and Sentinels or Cage Shaking

  • Advantages: Effective even when air filtration occurs at cage level before reaching exhaust ducts (Bauer, 2016; Dubelko, 2018). Can use in cages with sentinels or use cage shaking method (Dubelko, 2018). PCR testing of filter paper from cages shaken twice weekly was MORE effective than PCR testing of feces & fur from sentinel mice. Can test at 1 month except for very low prevalence organisms (e.g., Spironucleus).
  • Effective for: Pinworms & Fur Mites, regardless of type of bedding (Gerwin, 2016); MNV, Helicobacter, Rodentibacter spp., (better than sentinels), Entamoeba muris, Spironucleus spp. (Dubelko, 2018)
  • Challenges: Still reliant on soiled bedding transfer (amount of soiled bedding, dilution factor, frequency of transfer) & dependent on personnel shaking cages.

Removing testing altogether or testing individual animals via PCR

  • If animals from approved vendors will only be on-site for a short period of time (e.g., <1-2 months) then health monitoring may not be necessary.
  • Can consider collecting fecal samples, fur swabs +/- oral swabs directly from colony animals.
Common Concerns with Switching to Environmental Monitoring
  • Is there really enough data on this? More and more data is coming out (see references below). So far we have about 7 years of good data and success.
  • It's too costly! In fact, in some cases, especially considering the cost of housing & caring for sentinel rodents, it may be less expensive to conduct environmental monitoring (Luchins, 2020).
  • I don't have time to retrain my staff & develop a new program. Although there may be an initial time investment, there are time savings once implemented (Luchins, 2020).
  • Will other institutions accept our rodents? Reports from institutions currently using only environmental monitoring indicate that yes, other institutions will accept their rodents. In fact, other institutions recognize the increased sensitivity of this type of health monitoring program.
  • Will there be false positives or ambiguities? This is possible. Always investigate unexpected results further.
  • Will there be residual nucleic acid after rack washing? There may be some for Helicobacter spp. or MNV though may not (Mailhot, 2019). If seen, you may need to wash racks more than once or even scrub plenums to remove residual nucleic acids.
  • What about missing new & emerging pathogens? This problem can be helped by performing histopathology on any colony animals with unusual phenotypes/signs/illness.
How common is environmental monitoring?
  • In a webinar survey of 172 individuals, 79% say their institution use some form of environmental monitoring.
  • In a webinar survey of 167 individuals of 82% say they are still using sentinel animals.
What should I do if I find something?
  • This may depend on the pathogen. Ask your diagnostic laboratory what they recommend. They may recommend:
    • Testing colony animals by cage perimeter swabs, direct fur swabs, blood, or feces to narrow down positive cages.
    • Pooled plenum swabbing for confirmatory testing
  •  If you suspect a false positive or residual nucleic acid, then move cages to a clean rack & re-swab in 2-4 weeks.
  • Consider submitting to a different diagnostic lab for confirmatory testing.
Further research is needed on implementing environmental monitoring with cage level filtration
  • Does filter paper need to be in the lid or can it be in direct contact with soiled bedding?
  • How much cage shaking is actually needed?
  • Testing with introduced pathogens would help confirm detection capabilities

On September 16, 2020 NA3RsC put together a panel webinar on Applications in Developing Technologies for Rodent Health Surveillance. 

Our speakers were Susan Dowling, DVM.; Christina Pettan-Brewer, DVM, MSC; Patricia L. Foley, DVM, DACLAM; and Chris Manuel, DVM, PhD, DACLAM.

To view a recording of our webinar, click here.

After participating in this webinar, attendees will be able to

  • Better understand the strengths and limitations of using the latest diagnostic technologies for rodent health surveillance
  • Make more informed decisions on how to use the latest diagnostic technologies in their health surveillance programs
  • Be able to guide and make recommendations to developers and suppliers for the advancement of technologies in this area.

We had over 694 individuals register for this webinar when it was live. We have received feedback from two large academic and government institutions that they will be changing over to full environment monitoring after viewing this webinar. Please share it with your networks so we can work together to reduce/replace sentinel animals.

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 Science55(6), 782-788.
  2. Besselsen, D. G., Wagner, A. M., & Loganbill, J. K. (2000). Effect of mouse strain and age on detection of mouse parvovirus 1 by use of serologic testing and polymerase chain reaction analysis. Comparative medicine50(5), 498-502.
  3. Brielmeier, M., Mahabir, E., Needham, J. R., Lengger, C., Wilhelm, P., & Schmidt, J. (2006). Microbiological monitoring of laboratory mice and biocontainment in individually ventilated cages: a field study. Laboratory animals40(3), 247-260.
  4. Compton, S. R., Homberger, F. R., Paturzo, F. X., & Clark, J. M. (2004). Efficacy of three microbiological monitoring methods in a ventilated cage rack. Comparative medicine54(4), 382-392.
  5. de Bruin, W. C. C., van de Ven, E. M. E., & Hooijmans, C. R. (2016). Efficacy of soiled bedding transfer for transmission of mouse and rat infections to sentinels: a systematic review. PloS one11(8), e0158410.
  6. Dole, V. S., Zaias, J., Kyricopoulos-Cleasby, D. M., Banu, L. A., Waterman, L. L., Sanders, K., & Henderson, K. S. (2011). Comparison of traditional and PCR methods during screening for and confirmation of Aspiculuris tetraptera in a mouse facility. Journal of the American Association for Laboratory Animal Science50(6), 904-909.
  7. Dubelko, A. R., Zuwannin, M., McIntee, S. C., Livingston, R. S., & Foley, P. L. (2018). PCR Testing of Filter Material from IVC Lids for Microbial Monitoring of Mouse Colonies. Journal of the American Association for Laboratory Animal Science57(5), 477-482.
  8. Gerwin, P. M., Ricart Arbona, R. J., Riedel, E. R., Henderson, K. S., & Lipman, N. S. (2017). PCR testing of IVC filter tops as a method for detecting murine pinworms and fur mites. Journal of the American Association for Laboratory Animal Science56(6), 752-761.
  9. Ike, F., Bourgade, F., Ohsawa, K., Sato, H., Morikawa, S., Saijo, M., ... & Berard, M. (2007). Lymphocytic choriomeningitis infection undetected by dirty-bedding sentinel monitoring and revealed after embryo transfer of an inbred strain derived from wild mice. Comparative medicine57(3), 272-281.
  10. Jensen, E. S., Allen, K. P., Henderson, K. S., Szabo, A., & Thulin, J. D. (2013). PCR testing of a ventilated caging system to detect murine fur mites. Journal of the American Association for Laboratory Animal Science52(1), 28-33.
  11. Kapoor, P., Hayes, Y. O., Jarrell, L. T., Bellinger, D. A., Thomas, R. D., Lawson, G. W., ... & Nielsen, J. N. (2017). Evaluation of anthelmintic resistance and exhaust air dust PCR as a diagnostic tool in mice enzootically infected with Aspiculuris tetraptera. Journal of the American Association for Laboratory Animal Science56(3), 273-289.
  12. Körner, C., Miller, M., & Brielmeier, M. (2019). Detection of Murine Astrovirus and Myocoptes musculinus in individually ventilated caging systems: Investigations to expose suitable detection methods for routine hygienic monitoring. PloS one14(8), e0221118.
  13. Leblanc, M., Berry, K., Graciano, S., Becker, B., & Reuter, J. D. (2014). False-positive results after environmental pinworm PCR testing due to rhabditid nematodes in corncob bedding. Journal of the American Association for Laboratory Animal Science53(6), 717-724.
  14. Lindstrom, K. E., Carbone, L. G., Kellar, D. E., Mayorga, M. S., & Wilkerson, J. D. (2011). Soiled bedding sentinels for the detection of fur mites in mice. Journal of the American Association for Laboratory Animal Science50(1), 54-60.
  15. Luchins, K. R., Bowers, C. J., Mailhiot, D., Theriault, B. R., & Langan, G. P. (2020). Cost Comparison of Rodent Soiled Bedding Sentinel and Exhaust Air Dust Health-Monitoring Programs. Journal of the American Association for Laboratory Animal Science59(5), 508-511.
  16. Mahabir, E., Durand, S., Henderson, K. S., & Hardy, P. (2019). Comparison of two prevalent individually ventilated caging systems for detection of murine infectious agents via exhaust air particles. Laboratory animals53(1), 84-88.
  17. 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 Science59(1), 58-66.
  18. 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 Science55(1), 58-65.
  19. Manuel, C. A., Pugazhenthi, U., Spiegel, S. P., & Leszczynski, J. K. (2017). Detection and elimination of Corynebacterium bovis from barrier rooms by using an environmental sampling surveillance program. Journal of the American Association for Laboratory Animal Science56(2), 202-209.
  20. 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 Science55(6), 775-781.
  21. Miller, M., Ritter, B., Zorn, J., & Brielmeier, M. (2016). Exhaust air particle PCR detects Helicobacter hepaticus infections at low prevalence. J Vet Sci Technol7(343), 2.
  22. Miller, M., & Brielmeier, M. (2018). Environmental samples make soiled bedding sentinels dispensable for hygienic monitoring of IVC-reared mouse colonies. Laboratory animals52(3), 233-239.
  23. 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 Science59(2), 156-162.
  24. Perdue, K. A., Copeland, M. K., Karjala, Z., Cheng, L. I., Ward, J. M., & Elkins, W. R. (2008). Suboptimal ability of dirty-bedding sentinels to detect Spironucleus muris in a colony of mice with genetic manipulations of the adaptive immune system. Journal of the American Association for Laboratory Animal Science47(5), 10-17.
  25. 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 Science58(2), 208-215.
  26. Zorn, J., Ritter, B., Miller, M., Kraus, M., Northrup, E., & Brielmeier, M. (2017). Murine norovirus detection in the exhaust air of IVCs is more sensitive than serological analysis of soiled bedding sentinels. Laboratory animals51(3), 301-310.

If there are other health monitoring resources that you think should be featured on this page, please contact us at contactus@na3rsc.org.