Terminology

Accurately communicating objectives in an animal laboratory setting is vital, and therefore, so is a good understanding of the appropriate terminology. While terminology pertaining to managing animal colony health can sometimes be confusing, understanding the following key terms will help personnel to avoid miscommunications.

• Isolation: A broad term frequently utilized in the laboratory animal setting. It commonly refers to the separation of animals, personnel, and/or research materials to prevent cross-contamination of organisms and infectious agents between animals, spread of zoonotic agents, and contamination of the surrounding environment. Isolation practices can be broken down into multiple specific terms, depending on what type of isolation is required. Quarantine, biosafety, biosecurity, biocontainment, and bioexclusion are all related but distinct terms, and a careful animal program manager should avoid using them interchangeably.
• Quarantine: The isolation of animals possessing either unwanted or unknown health status.
• Biosafety: The practice of creating safe conditions in research environments by decreasing or eliminating the exposure risk of potentially hazardous biological agents to individuals and the environment (CDC 2009). This term is distinct from the others, as it typically relates to human health, not that of the animals.
• Biosecurity: This term has multiple definitions, depending on the discipline. Mainly, it describes the methods used to avert biological terrorism or other disease outbreaks. However, in an animal facility, the term can relate to the protection of an animal colony from contamination from known or unknown infections that may cause disease and/or lead to research variables (NRC 2011). Additionally, it relates to the protection from loss, theft, diversion, or intentional misuse of biological materials, microorganisms, and research-related information. Biosafety and biosecurity have somewhat different goals, but overall, they are generally complementary (CDC 2009; Nordmann 2010). Biosecurity practices can be further broken down into biocontainment and bioexclusion.
• Biocontainment: This is the specific process or engineering control that reduces the risk of external transmission and propagation of organisms that could be dangerous to human and/or animal health. It involves methods to prevent the accidental release of pathogenic organisms or agents, including bacteria, viruses, and biological toxins. Physical containment is utilized to avoid accidental occupational infections and/or release into the surrounding community.
• Bioexclusion: The measures taken to reduce the risk of introduction of pathogens into the animal facility. In terms of the animal colony, this involves defining a specific pathogen-free (SPF) status and the means utilized to maintain this status. This leads to the commonly used term barrier, which defines how a facility maintains a certain health status. Any unwanted pathogen in a facility is termed an adventitious agent.
• Barrier: A systematic and comprehensive program used for the prevention of pathogen introduction into the colony animals. Barriers vary widely in scope, and could relate to an individual cage or room, or an entire facility. They also consist of multiple elements, including the housing of animals with known specified pathogen status, a monitoring system to maintain this status, the design of the housing environment, and the management of the physical plant and caging environments.

Performing a Risk Assessment

While appropriate bioexclusion practices are the cornerstone of an effective animal research facility, it is simply not practicable to exclude all pathogens. Further, unnecessary isolation and containment practices in bioexclusion programs can increase costs and personnel workload, also potentially delaying research. For these reasons, a thorough risk assessment should be made for each pathogenic agent of concern that can accurately identify the costs and benefits of exclusion. The step-by-step risk assessment process below elucidates the potential hazards in your facility and their impact to the research program. This, in turn, will allow facility leaders to develop the necessary bioexclusion practices for the agents that pose a genuine risk to the program and research (White et al. 1998; CDC 2009).

Process and Participants Involved

Prior to beginning the risk assessment process, it is important to make sure that all the necessary participants are included in the discussion. In a small facility, this may include all staff members; in a larger facility, it may be best to have a small group of individuals with subject-specific knowledge gather the necessary information for a final risk assessment. The key participants to include are the veterinarian, facility manager, diagnostic lab representative, and principal investigator or laboratory director, who can speak to the impact of agents on their research.

To conduct a risk assessment for a bioexclusion program, first rank each microorganism based on its known potential to cause disease and potential research interactions. This can be broken down further by evaluating multiple factors and how those relate to the species utilized and the research performed. For a good step-by-step discussion of performing a risk assessment for a bioexclusion program, see White et al. (1998). After evaluation, divide the microorganisms considered into three categories of risk: low, medium, and high. These categories will help determine the necessary practices when working with these organisms (White et al. 1998).

Transmission of Agents

In determining the likelihood of an agent entering the animal colony, the risk assessment needs to consider transmission of the organisms. There are several methods by which organisms can be transmitted to other animals, and the measures required to control the agents will need to correspond to the transmission pathway. For example, organisms that are transmitted via aerosol”“in other words, through the spread of contaminated airborne droplets or dust”“require very strict control measures to prevent spread (Shek et al. 2015). Agents may also be spread by direct contact or oral ingestion, which tend to be easier to control. Insect vectors can also be responsible for transmission of agents between animals in a facility; however, if pest control is adequate, these types of agents should be of minimal concern. A thorough review of routes of transmission is available from the Centers for Disease Control and Prevention (CDC 2009) and Whary et al. (2015).

Establishing Standards for an Animal Care and Use Program

After a thorough risk assessment, each program must establish its own standards for bioexclusion and how they will be implemented at the facility. The establishment of these standards should involve discussion with multiple individuals within the program, including facility managers, veterinarians, animal researchers, and possibly institutional oversight committees, such as the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). Once established, these standards should be reviewed periodically to ensure that they are still meeting the needs of the program and, where appropriate, adapt to the fluctuating needs of the research, other programmatic adjustments, and the discovery of new adventitious agents.

Defining SPF Criteria

SPF refers to an animal that is free of a list of pathogens, but otherwise has an undefined microflora (Rahija 2007). The term SPF alone has little meaning, as facilities often differ in the adventitious infections and parasites allowed to enter. Therefore, a critical component of any bioexclusion program is defining which adventitious infections and parasites are of concern to the facility and the steps that will be taken to prevent their entry.

The determination of whether an agent will appear on the SPF list is influenced by several factors, including, but not limited to, the potential interference of the agents with research, the ability to cause clinical disease, the need to transfer animals between facilities, and the control mechanisms needed to exclude the agents. The Federation of European Laboratory Animal Science Associations (FELASA) has developed recommendations for which infectious agents to monitor and the frequency that monitoring should be performed for mice, rats, hamsters, guinea pigs, and rabbits (Mahler Convenor et al. 2014), and this document can be helpful when developing an exclusion list. Tables 33.1 and 33.2 provide common pathogens excluded from mouse and rat barrier facilities, respectively.

Table 33.1

Bacteria, Viruses, and Parasites That Are Commonly Excluded from a Mouse Barrier Facility.

Table 33.2

Bacteria, Viruses, and Parasites That Are Commonly Excluded from a Rat Barrier Facility.

In contrast to an SPF animal, a conventional animal is one that is raised in an environment that may have unknown microflora and may have an unknown disease status (Rahija 2007). These animals therefore do not necessarily need to be housed in barrier conditions. However, because unknown pathogen status can have unwanted results on research, most institutions are gradually phasing out the use of these types of colonies.

FELASA also has a working group for nonhuman primate health, which published suggestions for harmonized health management in 1998 (Weber et al. 1999). However, as these suggestions are in need of updating, more relevant publications exist that describe the SPF status for Macaque spp. (Morton et al. 2008). The need for SPF colonies is critical for preventing the spread of disease to human personnel, particularly as nonhuman primates harbor many zoonotic diseases (e.g., Mycobacterium tuberculosis and Macacine hervpesvirus 1). For this reason, SPF colonies usually exclude these organisms, in addition to simian immunodeficiency virus (SIV), simian type D retrovirus (SRV), and simian T cell lymphotropic/leukemia virus (STLV), as these present risks to investigators and can confound research results, especially for those studying SIV and AIDS (Morton et al. 2008).

For other animal species, health monitoring programs might not be as robust or organized; however, SPF criteria are still necessary to exclude adventitious pathogens. Restriction of these adventitious pathogens and parasites from the facility will increase research data reproducibility and reliability and decrease the risk of zoonotic transmission. FELASA has publications describing criteria for cats, dogs, and pigs (Rehbinder et al. 1998) and for small ruminants (calves, sheep, and goats) (Rehbinder et al. 2000). With the increase in zebrafish populations in biomedical research facilities, health monitoring of this species is evolving. The same principles of health monitoring apply; however, due to the aquatic environment of fish, water quality, water source, and facility design must be considered. The Zebrafish International Resource Center (ZIRC) has responded with a description of adequate health monitoring for zebrafish (Kent et al. 2009; ZIRC 2009). For all these species, a reliable SPF vendor is the first step to ensuring animals that are free of adventitious pathogens and parasites.

Facility Operational Methods

Once an SPF list has been created, the next step is to delineate the ideal control measures to maintain this level of bioexclusion in the animal colony. Control measures include vendor selection, shipping, isolation or quarantine, housing, facility design, transport, and use of the animals. These control measures are used in combination with the routine monitoring program to ensure that adventitious organisms are excluded from the facility (White et al. 1998).

Operation of facilities to maintain colony health can be separated into primary and secondary levels of preventing the introduction of unwanted agents. The primary level is the first line of defense in maintaining bioexclusion. Equipment such as ventilated caging racks and biosafety cabinets or change stations to change cages is part of this primary level, along with the use of pertinent personal protective equipment (PPE), which differs based on the animal species and potential for introduction of infectious agents or pathogens. PPE such as gloves, hair bonnets, respiratory protection, boot and shoe covers, and lab coats or disposable gowns is used for coverage of exposed body surfaces, for example, face, eyes, hands, and hair, as well as exterior clothes. Although the use of PPE is an important consideration, the amount needed should be evaluated for each facility. Recent studies have shown that some PPE components do not appreciably improve and, in some cases, actually compromise attempts to maintain bioexclusion (Hickman-Davis et al. 2012; Baker et al. 2014).

Secondary levels include the design and construction of the facility, such as physical separations of functional areas and engineering controls. Examples of physical separations include doors to limit entry, anterooms to don and remove PPE, and shower-in capabilities. Engineering controls may consist of heating, ventilation, and air-conditioning (HVAC) systems and waste treatment systems (King et al. 1999; CDC 2009).

Flexibility incorporated into facility design can accommodate the needs of an ever-changing research environment, which may permit the ability to house different species (nonhuman primates vs. rodents) and animals of varying pathogen status (aerosol transmission vs. direct contact). Determining the optimum room size that will accommodate multiple species will provide the facility manager with more flexibility to use the rooms for various uses and species. Room pressurization is also important in using rooms for bioexclusion or quarantine. A room that can be changed from positive pressure, for bioexclusion, or negative pressure, for quarantine, can greatly increase the ability to accommodate different needs.

Facility surfaces should be easily sanitizable and decontaminated. Additionally, all floors, walls, and ceiling seams should be sealed to prevent incursions by feral rodents and insects. Foresight is necessary when designing a facility, as this will allow the institution to prepare for future needs. Specific information about facility design can be found in Chapter 18.

Species Differences

Bioexclusion control practices differ with the species utilized in the facility. For mice, rats, and other rodents, the control of adventitious organisms in most facilities occurs at the cage level, as these animals are housed in static microisolators or individually ventilated cages. The ventilated caging systems frequently provide high-efficiency particulate air (HEPA)”“HEPA filters exclude 99.97% of particles that have a size of 0.3 μm”“to each individual cage and, depending on the type of ventilated caging, may also have a HEPA filter for the air leaving the cage. For a bioexclusion program, it is preferable to house rodents in a positively pressurized cage to prevent entry of adventitious agents into the cage. Room pressurization, as discussed above, should also be considered, depending on the agents of concern, the effectiveness of the caging system at isolation, and how the animals will be managed within the room.

While all caging needs to be sanitized, the bedding, feed, water, and enrichment may need to be sterilized, depending on the level of the bioexclusion desired. This needs to be determined based on which agents are to be excluded, the health status of the animals (immunodeficient vs. immunocompetent), and the probability of the agent entering the colony via these routes.

For rodent facilities, anytime a cage needs to be opened”“irrespective of whether it is for research procedures or cage change”“there is a risk of compromising bioexclusion. This risk is best mitigated by handling the cage only in a Class II biological safety cabinet, laminar flow hood, or cage change station, and using an appropriate disinfectant agent on the cage and gloves of the handler. These different aspects of the animal program will need to be considered in the risk assessment process.

For larger species, such as dogs, pigs, and nonhuman primates, the bioexclusion control operates at the room level because the housing occurs in open caging or runs where contact between animals from different cages is possible. To maintain bioexclusion, air pressure differentials are set up between rooms and corridors. The building HVAC system is also utilized by controlling airflow at the facility level (e.g., moving air from the cleanest room in the barrier [surgery suite or clean cage wash] to the dirtiest room [necropsy or dirty cage wash]). In addition, cubicles can be used to isolate different groups of large animals in a single defined control area. These cubicles are each set at a negative air pressure to the room so as to ensure no cross-contamination of air between the groups of animals. However, once the door to the cubicle is open, the differential ceases to exist as it equates with the room pressure, and so only one cubicle door per room should be open at a time (White et al. 1998).

Outbreak Control

Prevention of adventitious pathogens is the key to a good bioexclusion program; however, if an unwanted adventitious pathogen or parasite is detected during colony health surveillance, the manager should address the situation quickly to decrease the chance of spread to the remainder of the colony. The urgency to address the breach and the decontamination process available will depend on the transmission characteristics of the agent detected. Additional information about specific agents can be found in Brayton et al. (2004), Clifford and Pritchett-Corning (2012), and Pritchett-Corning and Clifford (2012).

Initially, confirmation testing by a different testing method than originally utilized or other laboratory should be considered. If the positive test was found in a sentinel animal, the organism may no longer be detected in the research colony due to the time delay in health monitoring. Therefore, it can be difficult to identify the index animal. Once the pathogen has been confirmed, the suspected positive animals or room should be isolated so as to decrease the potential spread to other animals in the facility. Isolation can be established within the room, or animals can be relocated to a remote facility or room. If isolation is established in the room, modifications to the building or room function and procedural changes should be considered. If possible, room pressurization should be changed from bioexclusion to quarantine to prevent possible spread at the room level. Additional containment practices should all be considered to prevent spread of the agent as a fomite or directly on a contaminated animal. These include restricting personnel entry, limiting animal movement into and out of the room, and procedural measures, such as bagging and decontaminating dirty supplies out of the room. The procedures implemented will depend on the transmission characteristics of the agent and continued access to the animals in the room.

Once isolation has been established, the facility leadership will need to determine how to resolve the situation. Once the agent has been confirmed, the options are usually to treat, cull, or rederive the animals. The same group of individuals that participated in the risk assessment process and in the creation of the SPF criteria can be assembled to develop a plan for returning the room to SPF status. This group can help to balance the animal and research needs of the facility.

If treatment options are available, these can be pursued as long as they are feasible and will not affect the research at hand. After an adequate course of treatment, testing must be redone to ensure negative results and elimination of the organism. Some organisms can be “burnt out” by halting breeding and introductions of new animals to the colony. This option is limited to those agents that either do not persist in the environment or transmit via placental transfer. Culling the whole facility or room may be required for select organisms when this is the only acceptable method (e.g., zoonotic organisms). Culling the affected animals also may be the most efficient option when the postcontamination research value is limited and the animals are readily available from a commercial vendor. Rederivation, either by cross-fostering or embryo transfer, is the mechanism most commonly used for rodents; however, it requires technical proficiency, time, and ample funds (White et al. 1998).

Biologics

Besides live animals, biological materials, including blood products, cell lines, and other tissues derived from or passaged in rodents, are also transferred frequently between institutions. Once these biological agents are introduced into living mice, they become potential vehicles for pathogens. Consequently, pathogen testing of biologics must be done before they are introduced into live animals in the barrier facilities. This type of testing is provided by many commercial vendors using PCR technology. There has to be a fine balance between the frequency and requirement of testing so as to avoid unnecessary costs while still ensuring that the animals maintain their SPF status. As such, a risk assessment should also be completed for use of these types of materials. This type of assessment is detailed in the Peterson article (Peterson 2008).

Animal Import

Approved commercial vendors should be identified that have the same or superior health status as the barrier colony animals. However, researchers may need to use animals from nonapproved vendors, such as other biomedical research institutions. Depending on their health status, when these animals are imported into your facility, they may need to enter an import quarantine. As the exclusion list varies widely between different institutions, this decision to quarantine or not can be a difficult one. Therefore, FELASA has developed a tool where “the main objective ” is to harmonize health monitoring (HM) programs (i.e., designing, sampling, monitoring, reporting and interpreting) which will help to improve knowledge about the microbiological quality of animals used in research and to meet scientific, legal, and welfare requirements” (Mahler Convenor et al. 2014). In addition, a FELASA–American Association for Laboratory Animal Science (AALAS) joint working group has been established to determine whether a common health report can be utilized for international transfers. This group has developed a customizable spreadsheet for health report formatting that can be used to exchange this information (Pritchett-Corning et al. 2014).

Once the health status of the new shipments can be ascertained, the suitability of these animals for entry into the colony must be determined. This is often done by comparing the quarantine results to established health criteria (i.e., the SPF list). Further discussion regarding evaluating and maintaining health status can be found earlier in this chapter, as well as in other references (Rehg and Toth 1998; Lipman and Homberger 2003; Roberts and Andrews 2008).

If the health statuses do not match, an import quarantine can be used to determine the pathogen status of newly arrived animals to determine if and when they can enter the colony and what actions might be needed to allow for their entry. Therefore, newly arrived animals are commonly isolated from colony animals during a quarantine period to evaluate for evidence of disease and infectious agents that are excluded from the current colony. To protect the existing colonies, the imported animals are usually placed in containment at the cage level or the room level, depending on the species and risk. Rodents are often tested or prophylactically treated for parasites (e.g., pinworms and fur mites) and tested for evidence of adventitious agents by serology, parasitology, and/or PCR. Larger animals may be tested and treated for common bacterial and parasitic infections and vaccinated if necessary. Nonhuman primates screening varies by species, but typically includes an evaluation for tuberculosis, and possible viral (e.g., Macacine herpesvirus 1 and retroviruses), bacterial (e.g., Shigella sp.), and parasitic agents. A more detailed discussion regarding primate import quarantine is available elsewhere (Kramer et al. 2012).

One refinement to the practice of live animal import quarantine, used primarily for rodents, involves the importation of cryopreserved embryos or sperm. This alleviates the need to ship live animals, which reduces stress to the animals and potential contamination during shipment. Rederivation using embryo and/or sperm, however, does not eliminate all adventitious pathogen concerns, as contamination of these tissues with mouse parvovirus (Agca et al. 2007) and endogenous ecotropic murine leukemia viruses (Hesse et al. 1999) has been documented. However, some agents, including ecto- and endoparasites, are eliminated completely, and the risk of others is greatly reduced. The testing of these tissues must be similar to the testing of biological material for contaminants (described above), which is likely to be less involved and lower in cost (Kelley 2010).

Isolation

When a few cages of rodents need to be isolated due to a disease outbreak or other situation that results in their health status being questioned, isolators can be used instead of standard caging. Many styles of isolators exist, but the most common for rodents are the semirigid and flexible film isolators. All equipment and materials entering the isolator are disinfected or sterilized to ensure that they are free of adventitious microorganisms. Isolators, which have air HEPA filtered in and out, provide bioexclusion properties provided there is no break or tear in the isolator. The use of isolators adds a lot of flexibility to a bioexclusion program (White et al. 1998).

With the increase in technological advances in research techniques and equipment, such as imaging, there has been an increase in demand for temporarily housing animals from other institutions for very short durations (e.g., 1–2 days). While some of the animals could go through the import quarantine process, some studies are time sensitive, and therefore would be invalidated if the animals were required to be quarantined. Institutions should consider how to accommodate these studies and what pathogen testing methods are necessary to protect the animal colony from adventitious agents. A containment room with the appropriate controls in place can be beneficial, as this will allow research procedures that may not usually be performed. It is important to consider how these animals of unconfirmed health status will be contained within the isolation room. Additionally, their transport and use in procedural equipment rooms, such as MRI or computed tomography (CT), will also need to be contained to prevent possible contamination of the equipment with an adventitious pathogen.

Special Considerations

Access Control

Controlling access to different areas of the program allows managers to segregate animal colonies based on animal health status, as described earlier in this chapter. Access control involves several methods to segregate different areas within a facility, including signage, physical controls (lock-and-key systems, electronic door cards, and retina and fingerprint scanners), and security personnel. This allows access only to personnel who are properly trained and have completed the necessary medical clearance, prevents the spread of adventitious pathogens, and restricts unwanted entry from individuals not involved in the animal care program. Electronic control systems are also advantageous, as they provide a record of time and location of all entries and exits from the facility. The complexity of the security design depends on the size of the facility, location (city vs. rural), and type of research being conducted (NRC 2011). Security can be enhanced by providing video cameras that record entries, exits, and activities of personnel within the facility.

SOPs and Policies

Standard operating procedures (SOPs) are essential for maintaining bioexclusion in a laboratory animal research facility. All procedures”“from how to quarantine import animals to regular cage change operations in a barrier facility”“should be standardized to ensure uniformity. Step-by-step directions should be written by the personnel who are actually conducting the procedures to ensure that operating procedures are clear and in accordance with best practices. An active SOP program requires new personnel to be trained on SOPs, periodic revision of the SOPs and retraining, and development of new SOPs as necessary. The National Institutes of Health (NIH) FAQ website recommends that “SOPs should be reviewed by the IACUC at appropriate intervals (at least once every three years) to ensure they are up-to-date and accurate” (OLAW 2015). As SOPs are required to ensure regulatory compliance, the IACUC is responsible for oversight of the program and evaluating the effectiveness (NRC 2011). It is essential to have the SOPs readily available to all personnel, possibly including research investigators to ensure compliance. An electronic system is recommended, as it allows controlled access and the ability to easily update when revisions are necessary.

In an outbreak situation, when an adventitious pathogen has been detected, SOPs become even more important as temporary methods are put in place that might modify the standard procedures. It is necessary to ensure that all personnel who enter the facility, from the personnel that grant access to the mechanical staff, are aware of the new procedures. Social media sources, such as departmental website blogs, can be a good tool to disseminate this type of information. This allows research investigators to ask questions and voice concerns if necessary. If the outbreak occurred due to a lapse in following an established SOP, the document should be critically reviewed to ensure accuracy and retraining should occur for compliance.

Training Program

SOPs are the building blocks of any training program. Therefore, it is imperative that the training on SOPs is adequate and refreshed periodically. The training program should be tweaked to provide the necessary guidance for the different personnel who enter an animal facility. Overall, all personnel who enter the research facility must be fully trained on all aspects of a bioresearch facility. Procedures to maintain bioexclusion”“such as how to don the proper PPE, follow the facility entry order, and perform decontamination procedures”“are especially important for maintaining the facility health status. All initial and refresher trainings need to be documented appropriately. An electronic format is ideal to document training, as it can be easily updated and multiple users can log in to check the progress of an individual’s training status.

Communication

Departmental websites are often the best method of communicating items such as the exclusion list and current SPF status of the colonies to the research staff. This allows the information to be utilized by the research staff at the institution, as well as external institutions that plan on receiving animals from your institution. If the institution has rooms with different pathogen statuses, this information should be clearly defined on the website. This list should be updated as changes are made to ensure that it is always up to date. Research staff should be familiar with the exclusion list, as there might be individuals that require a more stringent SPF status due to their research interests. For example, if a researcher is studying Staphylococcus aureus, he or she would not want to receive animals infected with this bacterium. Therefore, this information should be communicated to the personnel involved in animal ordering.