Guidance on Establishing Health Monitoring Panels for Laboratory Mice to Reflect Contemporary Pathogen Prevalence

Agents recommended for all HM programs.

Despite modern advances in colony management, many agents are still common and relevant to murine research colonies today and therefore warrant frequent testing. Some of the key factors contributing to their continued presence in research mice include persistent infection or colonization of the host, tolerance of agents by some institutions, the use of untreated husbandry materials,^14–16 pest incursions,^17,18 and agent characteristics that promote stability and transmission. Considering both prevalence and severity of disease (and thus high research impact if present), it is strongly recommended that mouse colonies be monitored for MHV, mouse parvoviruses (MPV/MVM), Theiler's murine encephalomyelitis virus (TMEV), EDIM, pinworms, and fur mites quarterly and excluded from all programs.¹⁰ Potentially lower detectable copy numbers in addition to lower prevalence and short/nonpersistent shedding of infectious virus in immunocompetent animals (as in the case of MHV and EDIM) create an increased potential for false-negative results. Were an infection to occur within a research colony, these agents can lead to costly research-related consequences.¹⁹ Agents having lower prevalences such as MAV, reovirus, PVM, and LCMV,¹⁰ but still the potential for substantial impacts on research colonies, should be tested for annually. Although many programs continue to test for and exclude ectromelia virus (mouse poxvirus) and Sendai virus, with a 0.00% prevalence from 2016 to 2020,¹⁰ eliminating these agents from routine HM panels in well-managed programs is recommended unless individual situational factors deem it necessary.

Establishing or maintaining a barrier facility for immunocompromised mice.

Often, a need develops for maintaining certain mouse colonies at a higher-level health status than the population of mice in a general colony. The development of a barrier room, suite, or facility presents a special opportunity to exclude adventitious agents that may influence specific research studies, protect vulnerable (for example, immunocompromised) or founder populations, maximize distribution options to facilities with differing exclusion lists, or to demonstrate a higher level of rigor for the sake of reproducibility. Barrier practices mitigate the introduction of agents from other lower-level heath status colonies that are sometimes housed adjacent to barrier spaces. As such, barrier facilities commonly house immunocompromised populations in which opportunistic agents should be monitored and/or excluded. Most programs do not exclude opportunistic bacteria as part of their routine HM program and instead reserve such monitoring for colonies under this higher-level health status. In addition to previously listed agents recommended for all HM programs, commonly excluded agents for immunocompromised animals may include rodent chaphamaparvovirus (RCHPV-1), Chlamydia muridarum, Corynebacterium bovis, Helicobacter spp., and Pneumocystis murina. Perhaps the most notable pathogen for this group is C. bovis. The introduction of C. bovis can be devastating, as it is easily transmitted among vulnerable mouse populations and readily contaminates the animal facility environment, which aids in infection perpetuation within a facility.²⁰ A fungal pathogen of note is P. murina, as it is ubiquitous, transmitted by aerosolization, and commonly found in immunocompetent or immunovague mouse populations. Although its reported prevalence is relatively low (<0.2%),¹⁰ this may be an underrepresentation due to unreliable sampling and testing methods (that is, SBS)⁹ and low frequency of screening among institutions. In the most vulnerable immunodeficient populations (such as NSG mice), P. murina can cause debilitating chronic progressive pneumonia. Lastly, the relatively new RCHPV-1 has been shown to cause nephropathy and tubulointerstitial fibrosis in immunocompromised mice, prompting its consideration for exclusion from these colonies as well.^21,22 Overall, the extent of exclusion within a barrier facility will depend on the program goals. As the number of excluded agents increases, there is an increase in surveillance costs and often a decrease in staff access and available sources of mice that can enter the facility. To protect this higher-level health status within a facility, we recommend testing for these agents based on risk assessment and at the program’s discretion.

New, emerging, and reemerging agents.

With the application of high-throughput genetic sequencing technologies, there is likely to be an increase in the number and rate of identification of agents that may be classified as opportunistic, pathogenic, or pests. Once identified, the influences on prevalence of these new, emerging, and reemerging agents will be multifactorial with emphasis placed on organism-specific transmissibility and vivarium-specific biosecurity practices. Noteworthy new, emerging, and reemerging agents include RCHPV-1,²² also referred to as mouse kidney parvovirus or murine chapparvovirus, which was identified in 2018; the published discovery of murine astrovirus 2 (MuAstV2) in 2020;²³ the emergence of Bordetella pseudohinzii between 2008 and 2016;^24,25 and the reemergence of C. muridarum in 2022 after first being identified in the 1930s.²⁶ The published prevalence of these agents based on samples submitted to international diagnostic laboratories are 10%,²⁷ “rare,”²⁸ 0.46%,¹⁰ and 16%,²⁶ respectively. The variability of prevalence of these contemporary agents demonstrates that agents that may be discovered in the future should not automatically be considered ubiquitous despite the lack of prior knowledge and unavailability of diagnostic testing.

Many factors will determine whether new, emerging, or reemerging agents are added to routine HM panels to simply monitor for presence of agents or ultimately be added to an institution’s exclusion list. Several of these factors are institutional, or even vivarium specific, and based on risk assessments. Deliberations may also occur at the national or international level, which may contribute to a collective movement toward exclusion. Two primary factors that can have a significant impact on the argument for excluding a new agent is the demonstration of adverse impacts on research outcomes and induction of clinical disease. This is demonstrated best by the 2003 identification of MNV.²⁹ While no longer considered new or emerging, MNV remains highly prevalent in research mouse colonies despite being identified >20 y ago.^10,30 MNV is not excluded by many research institutions because infection has limited impact on biomedical research and causes no clinical disease in most mouse strains.^30–33 A thorough assessment of each new, emerging, or reemerging agent will be required to determine whether screening or exclusion is appropriate or prudent. This process is currently underway for RCHPV-1, C. muridarum, and B. pseudohinzii. RCHPV-1 and C. muridarum are both widely distributed within research mouse colonies,^26,27 with a disproportionate clinical impact on immunodeficient mouse strains.^21,22,34,35 In contrast, B. pseudohinzii causes a subclinical infection in immunocompetent mice with limited data on its impact on immunodeficient mice. Despite a lack of clinical signs, B. pseudohinzii is known to elevate inflammatory cell counts in the lungs of infected mice, foreshadowing an impact on research outcomes.^24,36–38 Nevertheless, it could be argued that research has been successfully conducted for decades in the presence of these infections. Despite this, historical ignorance cannot be the sole rationale for continued tolerance. Rationale for continued tolerance may include high prevalence between, and within, colonies; difficulty of agent detection due to pathogen infectivity and shedding kinetics; and the significant cost and time that would be required to facilitate rederivation to eliminate these agents. Furthermore, exclusion at academic institutions may be dependent on the initial exclusion at commercial breeding facilities. For example, RCHPV-1–free mice were only recently available from most commercial vendors in 2022. In contrast, most mice from commercial vendors were found to be C. muridarum–free at the time of reemergence of this agent.²⁶ A collective movement will be needed within the scientific or laboratory animal science communities to add new, emerging, or reemerging agents to screening and ultimately exclusion lists at scale. Until that occurs, individual institutions will need to make risk assessments based on site-specific research programs and constraints to determine for which agents screening is conducted and those where testing is performed for exclusion.

Import, export, and quarantine.

The sharing of mice between institutions remains an important consideration when developing HM programs. Unlike with vendor-supplied SPF animals, research animal facilities typically require incoming animals from other institutions to undergo quarantine and testing upon arrival as this may be a point of entry for pathogens. In some cases, to preserve a higher health status, facilities may require that mouse lines are rederived before entry, but this is associated with significant costs and operational implications. The risk associated with introducing new mice should be evaluated for each transfer. This includes a review of HM reports specific to the animals being imported, an assessment of biosecurity practices, and a thorough review of the overall HM program of the exporting facility including history of outbreaks. Additional factors to consider may include the frequency and type of HM performed (for example, SBS, direct colony sampling, sentinel-free soiled bedding, exhaust dust testing), size/layout of facility and type of caging, the immune status and previous manipulations of the animals being transferred (for example, injections with biological materials), previous outbreaks, and the potential for contamination during the transfer. As part of a quarantine program, facilities may want to screen incoming animals with a more extensive panel of agents that includes those tested for on a regular basis by the routine HM program as well as agents of low prevalence such as Salmonella spp., Sendai virus, mousepox, hantavirus, polyomavirus, K virus, and mouse thymic virus (MTLV). This is because the importation of animals has high potential for introducing excluded pathogens to a facility. Screening of incoming animals may involve quarantine and testing animals directly or indirectly (that is, with environmental HM) via PCR and/or prophylactic treatment with antibiotic or antiparasitic medications.⁸

Facilities with specialized programs that regularly export mice to other institutions, such as model production or transgenic facilities/cores, may require a more extensive HM program with a larger number of agents excluded, compared with routine HM programs, to facilitate the transfer and acceptance of animals by receiving institutions without the need for further testing prior to export. Similarly, if a receiving facility has unique HM requirements, they may request that the exporting facility test the animals via direct colony sampling for specific agents (for example, hantaviruses) before exportation. These details are typically coordinated between the importing and exporting facilities as part of the transfer process.

Wild rodents and pests.

Preventing the introduction of wild rodents and pests is an important aspect of maintaining a research rodent animal care program free of adventitious agents. Animals that are not purpose bred for research (for example, pet shop animals, wildling mice, or wild rodents) can contain a wide array of potential pathogens.³⁹ If vermin incursions occur, or if mice from nontraditional sources are being used, additional testing of rodent colonies may be required to identify whether undesired agents were introduced and to control for any potential outbreaks. The HM panel may need to be adapted to ensure that agents commonly excluded from research rodents, but potentially present in wild rodents,¹⁸ are included in routine or temporary testing. In addition to routinely excluded agents, additional testing could include Sendai virus, hantavirus, Salmonella spp., Helicobacter spp. (H. bilis, H. ganmani, H. mastomyrinus, and H. typhlonius), mouse cytomegalovirus (MCMV), MNV, MTLV, and parasites. Outbreaks of the tropical rat mite, Ornithonyssus bacoti, are another common sequela to wild rodent incursions.¹⁷ As this mite is zoonotic and can cause immunologic research effects, early identification, treatment, and elimination are necessary.

Upon capture of a wild rodent, extensive testing of the animal (for example, by PCR and/or serology) should be performed. Most diagnostic laboratories will have a comprehensive panel that screens for all potential rodent agents (both historical and present) that can be used in this situation. Note, however, that some serology panels may not be compatible with wild rodents due to a lack of cross-reactivity with laboratory pathogen strains. If specific agents are detected in a captured wild rodent, these agents can be temporarily added to HM panels of potentially impacted colonies. For example, if a captured wild rodent tests positive for a previously nonmonitored agent, this agent can be added to the quarterly (or more frequent) screening of potentially affected colonies to prevent or mitigate the potential spread of infectious agent within the colony. Since many pathogenic agents can transmit through fomites (such as feed, bedding, or caging materials), enhanced environmental screening of potentially contaminated fomites may also be of benefit. Environmental HM can be readily used to identify these agents, which would not be normally detected using a SBS program.¹⁷ Facilities with a history or potential for wild rodent incursions, such as older buildings or those undergoing construction/renovation, as well as those where wild rodents are brought into the facility for research use, should consider adding O. bacoti and other wild rodent pathogens to their routine HM panels to promptly identify an outbreak.

Zoonoses.

Excluding zoonotic agents may be prudent for occupational health and safety. Fortunately, the prevalence and risk of zoonotic agents in modern research rodent colonies remain minimal. Although a pathogen of serious consequence, detection of hantavirus in laboratory mice has not been reported in at least the last 25 y according to the data collected and presented in pertinent publications.^2,4,10 Therefore, in a well-managed program, this pathogen may be removed from the routine HM panel. However, there is a risk of humans contracting hantavirus from wild rodents, so testing for hantavirus may be warranted with instances of wild rodent incursions. Also, since hantavirus is present worldwide, some facilities may choose to test for this pathogen when receiving animals from an international location. LCMV is also present worldwide and can result in serious illness in people with weakened immune systems. Historically, LCMV was a reported contaminant in one biological material,⁴⁰ but a recent publication reports a 0.00% prevalence in mouse serology samples and 0.02% prevalence in PCR samples from 2016 to 2020.¹⁰ Therefore, removal of this pathogen from routine HM panels may be merited, as well. Lastly, zoonotic ectoparasites such as tapeworms (for example, Hymenolepis nana) and mesostigmatid mites (for example, O. bacoti, Liponyssoides sanguineus, Laelaps echidnina) should be actively excluded from modern murine colonies. Exclusion can be achieved through effective management of imported animals and wild rodents/pests (for example, using custom HM panels), as previously discussed.

Biological materials.

The introduction of murine pathogens to a rodent colony through biological materials is a common concern for programs engaged in research that uses such materials.⁴¹ Pathogens that may be transmitted via this route include lactate dehydrogenase–elevating virus (LDV), MCMV, polyoma virus, M. pulmonis, and C. bovis. All except the latter have been shown to have nominal prevalence in murine samples analyzed by a commercial diagnostic laboratory from 2016 to 2020 (LDV <1.08%; MCMV <0.06%; polyoma <0.07%; M. pulmonis <0.09%).¹⁰ It is also important to consider that biological materials processed and stored decades ago may have a greater likelihood of harboring these undesirable pathogens. Since historical standards for exclusion were less stringent, and there was limited availability of PCR testing at the time for agents that are commonly excluded from research colonies today, biological materials that were produced in or passaged through historic animals may possess some of these undesirable pathogens. As is common in research, these aged samples may be retrieved from storage at any time and used in animals.⁴¹ An example is LDV which is still seen in modern facilities due to historical contamination of a basement membrane protein matrix used by researchers in solid tumor cancer research.⁴² Lastly, the introduction and dissemination of these agents will likely be devastating to animal colonies and certainly place a large financial burden on the institution for remediation and elimination. In recent times, this is particularly true for C. bovis, which is a relatively prevalent agent (2.93% by serology, 2.26% by PCR),¹⁰ and will clinically affect immunocompromised populations. Therefore, it is recommended that biological materials be screened for additional agents, beyond those screened for in a routine HM program, prior to use in research animals (via PCR on a portion of the biological material intended for inoculation).

Study-related exclusions.

Segmented filamentous bacteria (SFB) are spore-forming, commensal bacteria that populate the ilea of mice and other species, including humans.⁴³ SFB have attracted the attention of researchers in recent years as they are involved in the maturation and function of the host gut immune barrier.^43,44 Currently, no commercial vendors offer mice that are guaranteed to be free of this agent; however, one case report describes the use of ampicillin-medicated water to eradicate SFB from a mouse colony.⁴⁵ At some institutions, SFB may be excluded with selected areas being maintained free of the agent using restricted or entry order policies in combination with more frequent testing or through the use of isolator housing.

Other agents that institutions may want to exclude due to their effects on study-specific research may include MNV, Citrobacter rodentium, Salmonella spp., and trichomonads (for investigators focusing on gastrointestinal or immunologic research), Helicobacter spp. (for investigators focusing on gastrointestinal or hepatic research), RCHPV-1 (for investigators focusing on renal research), B. pseudohinzii, C. muridarum, Filobacterium rodentium, and P. murina (for investigators focusing on pulmonary research), and C. bovis (for investigators focusing on cancer research).⁴⁶ While it is beyond the scope of this work to provide a comprehensive review of research or clinical impacts of each agent that would provide relevance for exclusion, readers are referred elsewhere learn more about specific agents.⁴⁷