Research Models
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Ken Henderson, PhD
In Pathogen Screening, Your Rodent Infectious Agent Exclusion List is So 2010
With a diminishing prevalence of classic rodent agents, and a growing list of emerging agents, is it time to update your rodent pathogen screening list?
Imagine a grocery list where you continue to include the same exact items every week, but you also keep adding interesting new products to the list without cutting out items you are no longer eating. Unfortunately, that is exactly what we have done with our rodent infectious agent exclusion lists today.
As new emerging rodent pathogens have been added to our diagnostic pathogen screening lists, we continue to maintain the same list of rodent agents that are now quite rare or entirely absent. Furthermore, there is a growing tolerance for emerging pathogens since the cost of rederivation of transgenic lines for many research institutions would be too high to stomach, and this list of ignored agents is growing.
Laboratory rodent providers continue to routinely test for the prevalent, the uncommon, the novel, and the forgotten rare agents, because they need to consider the huge number of research vivaria depending on them to provide assured specific-pathogen free rodents. “Approved vendor” status is a term used to refer to rodent providers that maintain a rigorous program of rodent pathogen screening so that quarantine testing from these providers is not required by their institution.
Although most research vivaria permit direct entry of approved vendor-reared rodents into their facility without quarantine, some research entities maintain a program to send an unopened or immediately re-crated shipment of mice or rats directly to a diagnostic lab. Referred to as “vendor screening,” it provides assurances that an outbreak at a vendor involving an excluded rodent pathogen does not go undetected.
The amount of testing performed routinely at our CRL rodent production facilities has increased with the addition of new infectious agents. More importantly, by adding supplemental PCR environmental testing of our large production areas the frequency of testing has also increased. Therefore, it should not be surprising that in the last 20 years the unintentional introduction of an infectious agent in a rodent production facility is typically detected by internal QC testing before it is detected by research facilities with their own testing program. Given the tremendous amount of pathogen screening performed by rodent vendors, is it important for research facilities to continue to monitor for agents we have not seen in research facilities in decades?
Biosecurity improvements helped reduce some rodent pathogens
PCR technology, now a permanent and important tool for detection of rodent pathogens, can detect agents by environmental monitoring that go undetected by traditional soiled bedding sentinels. The use of PCR for direct testing of incoming mice in quarantine from collaborating research institutions has also reduced the number of unwanted guests brought into research vivaria.
It was likely the explosion of genetically engineered model creation and their distribution among institutions that continues to contribute to the sharing of infectious agents across these same research vivaria. There has been a positive outcome on the reduction of many historically excluded agents thanks to biosecurity improvements made over the last 20-30 years. This includes the routine use of decontaminating solutions, treated feed and bedding, and more vigorous pest management programs, all which have reduced the introduction and spread of rodent pathogens. Microisolator caging alone, which is designed to only permit filtered air into the cage, creates an environment where agents that are naturally cleared by the immune system are unable to effectively spread to other cages and propagate. By the time the presence of an agent on a microisolator rack is detected in soiled bedding sentinel rodents or PCR-based environmental testing, the agents often have already been naturally eliminated in the research rodents by their immune system.
Which rodent agents should we conduct pathogen screening for?
Now that there are well over 70 recognized rodent infectious agents to screen for these days, few
institutions have the financial resources to test for all of them routinely. Where should we direct our rodent pathogen screening resources? Some institutions focus on testing incoming animals by PCR during quarantine or simply rederive all incoming rodents by embryo rederivation before permitting entrance to assure no unwanted agents show up. Then for routine quarterly testing, these institutions limit the amount of routine screening by focusing on prevalent agents and agents most likely to enter their research vivarium through husbandry materials, research biologics, or wild rodents during quarterly testing. A slightly larger panel of agents can be used annually to screen for less common agents. Institutions that only receive approved vendor rodents, utilize good biosecurity practices, and have no known rodent incursion issues should consider a small prevalent panel for routine monitoring year-round.
The reality is that we all would care if a rare and impactful agent was detected in our rodent colonies. Therefore, many institutions are unwilling to reduce their routine pathogen screening list and others will continue the expectation that other institutions will continue to do so as well, and the use of large panels are perpetuated. However, a well-built exclusion list should use the detection of prevalent agents as a biological indicator. If a prevalent agent is detected, it could then trigger the use of a larger panel to see what else may have co-entered the facility as part of the biological breach.
What agents should be included in a short prevalent rodent pathogen screening panel?
A good place to start is to look for stable viruses which are the best indicators of a biosecurity failure. These viruses, which include rotavirus, parvoviruses, and picornaviruses, often show up after the failed decontamination of feed or bedding material due to an issue with the autoclave or incomplete gamma irradiation.
To cover agents likely to be associated with wild rodent incursions, it is best to look for lengthy or persistently shed agents that are prevalent and commonly transmitted via fecal pellets or even direct contact for some caging situations. These agents would include adenovirus, enteric rodent coronavirus, Helicobacter, pinworms, fur mites, and some protozoa. Except for fur mites, these agents also tend to transmit well to soiled bedding sentinels if the rack prevalence is beyond a few cages of shedding mice. If you accept murine norovirus in your research vivarium, and know you have positive mouse lines, you could consider it a good positive control for both sentinel and environmental testing, but at a low rack prevalence, detection may be inconsistent in soiled bedding sentinels. Protozoa have long been considered good biological indicators of a breach in biosecurity, but except for a handful of species such as Entamoeba, Chilomastix and Hexamastix, most do not readily show up in soiled bedding sentinels but can be detected by PCR-based environmental monitoring methods.
Highly immunodeficient mouse models pose a challenge in pathogen screening
Not only is the face of infectious agents evolving, but also the research models themselves. Another emerging challenge is the development of highly immunodeficient mouse models which may be impacted by opportunistic bacteria that we didn’t care about 25 years ago. Many of these bacteria can already be found within standard rodent strains, genetically engineered models, and even lesser immunodeficient mice that may not be impacted. However, extra precautions may be required for housing highly immunodeficient models as well as the use of extended pathogen screening panels to look for opportunistic bacteria, for which these models may have little to no defense for keeping them in check.
As with any exclusion list, a good place to start is knowing the potential impact of an infectious agent on the research being performed within a vivarium. If the agent is not prevalent or does not impact the research you are conducting, then these are good candidates for removal from your routine screening lists.
Lastly, don’t do this alone. If you are a scientist, reach out to your vivarium veterinarian for support, and if you are a veterinarian, reach out to your diagnostic lab to discuss your screening program and exclusion list with scientists and veterinarians that can help make good recommendations.