Role of Housing Modalities on Management and Surveillance Strategies for Adventitious Agents of Rodents. William R. Shek

Transcription

1 Role of Housing Modalities on Management and Surveillance Strategies for Adventitious Agents of Rodents William R. Shek Abstract Specific pathogen-free (SPF) rodents for modern biomedical research need to be free of pathogens and other infectious agents that may not produce disease but nevertheless cause research interference. To meet this need, rodents have been rederived to eliminate adventitious agents and then housed in room- to cage-level barrier systems to exclude microbial contaminants. Because barriers can and do fail, routine health monitoring (HM) is necessary to verify the SPF status of colonies. Testing without strict adherence to biosecurity practices, however, can lead to the inadvertent transfer of unrecognized, inapparent agents among institutions and colonies. Microisolation caging systems have become popular for housing SPF rodents because they are versatile and provide a highly effective cage-level barrier to the entry and spread of adventitious agents. But when a microisolation-caged colony is contaminated, the cage-level barrier impedes the spread of infection and so the prevalence of infection is often low, which increases the chance of missing a contamination and complicates the corroboration of unexpected positive findings. The expanding production of genetically engineered mutant (GEM) rodent strains at research institutions, where biosecurity practices vary and the risk of microbial contamination can be high, underscores the importance of accurate HM results in mitigating the risk of the introduction and spread of microbial contaminants with the exchange of mutant rodent strains among investigators and institutions. Key Words: barrier; biosecurity; health monitoring; microisolation cage; rederivation; rodents; sampling; sentinels The Evolution of Strategies to Eliminate, Exclude, and Detect Adventitious Infections Because they have a short life cycle, produce large numbers of offspring year-round, and show variation in inherited characteristics, mice and rats have been the principal experimental animal models for over 100 William R. Shek, DVM, PhD, is Senior Scientific Director of Research Animal Diagnostic Services at Charles River Laboratories in Wilmington, Massachusetts. Address correspondence and reprint requests to Dr. William R. Shek, Charles River Laboratories, 251 Ballardvale Street, Wilmington, MA or William.Shek@crl.com. years. In 1909, Clarence C. Little, who later founded the Jackson Laboratory, began selecting and inbreeding mice with specific coat colors to demonstrate that animals could be bred to fix inherited traits as Mendel had done with plants. The first inbred strains that Little and others developed were mainly used to study the effects of heritable factors on the development of cancer. In these early days, and for the next 40 years, the practices, equipment, and facilities for controlling adventitious infections of laboratory animals were rudimentary. For example, rodents were often housed in wooden cages. Highefficiency particulate air (HEPA 1 ) filtration, today so important for excluding and containing infections, was not developed until the 1940s (Cambridge Filter Corp. 1963). Standard veterinary approaches to controlling disease (e.g., improved sanitation and nutrition, antimicrobial treatments) were not sufficiently effective at excluding or controlling pathogens to prevent the frequent curtailment or confounding of studies due to disease (Mobraaten and Sharp 1999; Morse 2007; Weisbroth 1999). The technologies available for pathogen surveillance also were unsophisticated. It wasn t until the 1930s that the electron microscope was developed and the first virus seen with the human eye, and the late 1940s that scientists used electron microscopy to identify viruses (Nagler and Rake 1948). Cell culture for virus isolation and serology for viral antibodies were not commonplace until the late 1950s and 1960s (Hawkes 1979; Hayflick 1989; Parker et al. 1965; Rowe et al. 1959; Schmidt 1979). The discovery of the structure of DNA was not until 1953 (Watson and Crick 1953), and publication of the first report of the polymerase chain reaction (PCR 1 ) for in vitro sequence-specific biochemical replication of gene sequences was not until 1985 (Mullis 1990). After World War II, biomedical research expanded and incorporated more sophisticated and sensitive biological, analytical, and bioanalytical methods. These advances enabled the discovery of a variety of indigenous rodent viruses, often as contaminants of virus stocks and tumor cells. Although rodent infections with these viruses were typically asymptomatic, particularly in postweaning immunocompetent hosts, they nonetheless confounded experimental 1 Abbreviations used in this article: GEM, genetically engineered mutant; HEPA filtration, high-efficiency particulate air filtration; HM, health monitoring; MHV, mouse hepatitis virus; MNV, murine norovirus; MPV, mouse parvovirus; PCR, polymerase chain reaction; SPF, specific pathogen-free; VCS, ventilated (microisolator) caging system(s) 316 ILAR Journal

2 findings (Hartley and Rowe 1960; Kilham and Olivier 1959; Riley et al. 1960; Rowe et al. 1962). In addition, a few indigenous rodent viruses were found to be zoonotic agents that have been responsible for disease outbreaks in laboratory personnel exposed to silently infected cell lines or cellline-inoculated rodents (Baum et al. 1966; Bhatt et al. 1986; Hinman et al. 1975; Lewis et al. 1965; Lloyd and Jones 1986). Rederivation and Barrier Room Production As it became apparent that rodents suitable for research could not be produced by following standard veterinary disease control measures in the basic facilities that existed, beginning in the 1950s governmental, academic, and professional organizations helped to develop and promote best practices and to sponsor training and research in laboratory animal medicine (Allen 1999). Novel biosecurity systems were developed and instituted to eliminate pathogens and provide a barrier to their entry. Foster (1958) and others reported using hysterectomy derivation of rodents to eliminate horizontally transmitted pathogens. In this process, the gravid uterus from a dam in a contaminated colony was pulled through a disinfectant solution into a sterile flexiblefilm isolator, where the pups were removed from the uterus and suckled on axenic (i.e., germ-free) foster dams. After being mated to expand their number and associated with a cocktail of nonpathogenic bacteria to prime their immune system, rederived rodents were transferred to so-called barrier rooms for large-scale production. The room-level barrier to infection entailed HEPA filtration of incoming air, disinfection of room equipment and supplies, and limited access to trained and properly gowned personnel (Dubos and Schaedler 1960; Foster 1980; Schaedler and Orcutt 1983; Trexler and Orcutt 1999). Axenic and associated animals are classified as gnotobiotic, meaning that they have a known, or completely defined, microflora. Rodents produced in barrier rooms in uncovered cages are not gnotobiotic because of their exposure to microorganisms both in the environment and harbored by people. Thus they develop a complex microflora, although they may be referred to as specific pathogen-free (SPF 1 ) to indicate that the colony from which they originated tested negative for certain pathogens and perhaps other adventitious agents that may interfere with research without causing disease. For mice and rats, SPF refers to animals that are free of most exogenous viruses and other pathogenic microorganisms. It is noteworthy that although barrier room production had become standard practice for commercial rodent breeders by the1970s, a considerable percentage of commercial sources were still reported to be positive for a variety of adventitious viruses and parasites into the early 1980s (Casebolt et al. 1988). Emergence of Cage-Level Barriers When I began working in rodent diagnostics in the early 1980s, it was apparent that many research institutions were not prepared to maintain the health status of rodents, as SPF rodents from vendors often became ill or seropositive shortly after being received. Research institutions addressed this problem by implementing several strategies to exclude adventitious agents: the use of limited access barrier facilities, like those of commercial vendors; HEPA-filtered mass air cubicles and racks (McGarrity and Coriell 1976; White et al. 1983); isolators; and filter-covered cages. Although Kraft in 1958 demonstrated the effectiveness of the latter cage-level barrier strategy in preventing the spread of mouse rotavirus (Kraft 1958), and filter bonnets controlled the spread of infection in the 1960s and 1970s (Flynn 1968; Kraft et al. 1964), it wasn t until the 1980s that modern filter-top microisolation caging systems became commercially available (Hessler 1999; Sedlacek and Mason 1977; Trexler and Orcutt 1999). At first, microisolation cages were static (i.e., passively ventilated). Because the temperature, humidity level, and concentration of gases (such as CO 2 and NH 3 ) in static microisolation cages were significantly elevated compared to room levels, ventilated caging systems (VCS 1 ), pioneered by Edwin P. Les of the Jackson Laboratory in the 1960s (Les 1983), were developed to improve the microenvironment of rodents in filter-covered cages. VCS can also improve the room environment by reducing the production of NH 3 and by being exhausted directly into the facility HVAC. The direction of airflow in VCS can be changed to provide HEPA filtration of the air entering or exhausted from cages for the exclusion or containment of adventitious agents, respectively (Hessler 1999; Lipman 1999). Microisolation caging systems are often located in barrier rooms to provide a further obstacle to microbial contamination. Although flexible film and semirigid isolators provide the most dependable barrier to microbial contaminants, microisolation cages have become the dominant housing modality for SPF rodents, particularly in high-traffic research facilities, because in comparison to isolators they require much less space per animal. In addition, they are generally perceived to offer investigators easier access to their study animals (Hessler 1999). Transfer of Immunodeficient Rodent Colonies to Isolators to Exclude Opportunistic Pathogens As noted, rodents housed in barrier rooms in uncovered cages develop a complex microflora that includes microbes derived from the environment and harbored by people. Some of these microorganisms (e.g., Pseudomonas aeruginosa, hemolytic streptococci, Staphylococcus aureus, and Pneumocystis carinii) are not considered primary pathogens but rather opportunists because of their high propensity to cause disease in immunocompromised hosts, whether (1) immunosuppressed by irradiation or chemotherapy (Cryz et al. 1983; Flynn 1963a,b; Homberger et al. 1993; Weisbroth et al. 1998, 1999) or (2) innately immunodeficient, such as Volume 49, Number

3 athymic nude and severe combined immunodeficient (SCID) mice (Bosma et al. 1983; Clifford 2001; Clifford et al. 1995; Pantelouris 1968; Roths et al. 1990; Ward et al. 1996). Because it is not possible to reliably exclude many of these opportunists from rodents raised in uncovered cages (even in barrier rooms) (Blackmore and Francis 1970; Geistfeld et al. 1998), starting in the mid-1980s commercial suppliers moved the breeding of nude mice and other immunodeficient models to isolators and microisolation cages, which can effectively exclude opportunists if supplies are adequately disinfected and microisolation cages are opened only in laminar flow change stations or biological safety cabinets (Hessler 1999; Sedlacek and Mason 1977; Trexler and Orcutt 1999). Advances in Biotechnology and Diagnostics That Have Increased Demand for SPF Rodents and Enabled the Discovery of Additional Adventitious Agents In the 1980s, the nascent biotechnology industry applied advances in molecular genetics, immunology, and cell biology to the development and manufacture of modern biopharmaceuticals. A paramount requirement for the purity and safety of these products, and of the reagents and cell substrates used in their manufacture, is that they be free of extraneous microbes. Many biopharmaceuticals, such as monoclonal antibodies and recombinant proteins, are rodent-derived or generated in rodent cell substrates, a development that has accentuated the importance of excluding adventitious agents (particularly viruses) from rodent colonies to prevent them from contaminating cell substrates and reagents for eventual use in the manufacture of biologics (Shek 2007). Another significant outcome of advances in biotechnology has been the ability to produce in the laboratory transgenic and targeted knockout and knockin genetically engineered mutant (GEM 1 ) animal models, the vast majority of which are mice. Rigorous microbiologic quality control is particularly important for GEM colonies because an adventitious infection can alter a GEM strain s phenotype in a manner that results in spurious experimental findings. In addition, immunodeficiencies and other changes produced intentionally or unexpectedly by a genetic modification can increase the severity and alter the pathogenesis of infectious diseases (Franklin 2006; Karst et al. 2003; Pullium et al. 2003). Thus it is important to maintain GEM models as if they were immunodeficient: in isolators or microisolation cages and with disinfected supplies in order to exclude opportunists as well as primary pathogens. As demand for SPF rodents has increased, the use of new and more sensitive serologic assays (Smith 1986) and the introduction of molecular genetic techniques, most importantly the PCR (Compton and Riley 2001), have contributed to the discovery and characterization of infectious agents that are ubiquitous in rodent populations presumed to be SPF. For instance, the switch for parvovirus serology from the selective (i.e., virus-strain specific) hemagglutination inhibition (HAI) test to the highly sensitive enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA) provided serologic support for the existence of parvovirus serotypes besides the prototypical ones minute virus of mice (MVM), rat virus (RV), and H-1. This serologic evidence was substantiated by the discovery and characterization of several mouse parvovirus (MPV 1 ) serotypes, rat parvovirus (RPV), and rat minute virus (RMV). Researchers have shown that MPV-1 and RPV are nonpathogenic even for neonatal and immunodeficient animals (Ball-Goodrich et al. 1998; Smith et al. 1993), suggesting that they have been indigenous to mice and rats for a long time and are not emerging or postindigenous viruses. In addition, retrospective serology indicates that MPV was prevalent in mouse colonies more than 40 years ago (Jacoby et al. 1996). Although MPV and RPV are not pathogens, there are reports of their disrupting research by infecting proliferating lymphocytes and tumor cells (Ball-Goodrich et al. 1998; Jacoby et al. 1995; McKisic et al. 1993, 1998). Other recently identified rodent pathogens of note are the enterohepatic helicobacters (Fox et al. 1994; Ward et al. 1994) and murine norovirus (MNV 1 ) (Karst et al. 2003). Like the novel rodent parvoviruses, reports show that the helicobacters and MNV appear to have long-standing, highly adapted relationships with their rodent hosts, as they are prevalent in otherwise SPF rodent colonies (Hsu et al. 2006; Shames et al. 1995) and cause disease mainly or only in profoundly immunodeficient animals. MNV infections of GEM mice lacking innate immunity are pathogenic, whereas infections of other mouse strains are inapparent (Karst et al. 2003; Ward et al. 2006). The discovery of MNV therefore depended on recent progress in biotechnology, including the advent of GEM mice and sophisticated techniques for microbial genetic analysis. An additional shared characteristic of the parvoviruses, helicobacters, and MNV is that they are fastidious (i.e., not easily propagated in culture). For instance, of the various MPV strains that have been identified, only MPV-1a is cultivable and only in a mouse T lymphocyte clone (McKisic et al. 1993). Uniquely among noroviruses, MNV can be propagated in vitro, but only in a mouse macrophage cell line (Wobus et al. 2004). Helicobacter cultivation requires a microaerophilic atmosphere as well as fecal specimen filtration and special media supplemented with antibiotics to prevent overgrowth of other enteric bacteria (Fox et al. 1994). Fastidious culture requirements (and low pathogenicity) probably contributed to the comparatively late discovery of these agents but have not impeded their characterization or the development of diagnostic tests for them. This is largely because the traditional importance of microbial cultivation has been diminished (1) by rapid in vitro biochemical amplification of microbial genomic sequences using the PCR and (2) by the production of serologic antigens as recombinant proteins expressed from 318 ILAR Journal

4 microbial genes cloned into bacterial plasmid and baculovirus vectors (Ball-Goodrich et al. 2002; Compton et al. 2004a; Homberger et al. 1995; Riley et al. 1996). Biosecurity and Health Monitoring to Ensure the Exclusion of Known and Unrecognized Agents As discussed, SPF rodents for modern biomedical research need to be free of pathogens and other infectious agents that may confound research without causing disease. To meet this need, rodents have been rederived to eliminate adventitious agents and maintained behind barriers to exclude microbial contaminants. But a barrier may be breached due to its intrinsic shortcomings, equipment failure, or operator noncompliance with standard operating procedures, and therefore laboratory testing, referred to as health monitoring (HM 1 ), is necessary to verify that animals meet established SPF specifications (Livingston and Riley 2003; Shek and Gaertner 2002; Weisbroth et al. 1998). However, relying on laboratory testing to confirm the SPF status of an animal population has substantial limitations. False negative test results can occur because of inadequate assay sensitivity, analyst error, or sample selection error (e.g., submitting serum samples from SCID mice for serology). In addition, specific assay methods such as PCR and serology will probably miss inapparent infections with unrecognized agents; parvoviruses, helicobacters, and MNV are the most recent examples of agents that were inadvertently transferred among colonies and institutions before their discovery. Our experience at Charles River Laboratories has been that isolator-maintained rodent colonies rederived before the discovery of these agents have been uniformly free of them (as have most barrier room colonies stocked directly from isolators). For these reasons, effective microbiologic quality control for SPF rodents requires both strict adherence to biosecurity processes to eliminate and exclude adventitious agents, including those yet to be identified, and routine and comprehensive health monitoring. Neither biosecurity nor HM alone suffices. Effect of Housing Modalities on Sampling for Health Monitoring Accurate, meaningful health monitoring results require the sampling of an adequate number of appropriate animals on a sufficiently frequent basis. Animal Type and Modes of Exposure Colony Rodents for Commercial Barrier Room Production For large rodent production colonies housed in uncovered cages, animals for monitoring are selected from among breeder and stock rodents of various ages and sexes to enhance the likelihood of finding infections or positive assay results with an age- or sex-dependent (Thomas et al. 2007) distribution. During the production processes of mating and weekly regrouping of stock by age, sex, and weight, rodents are routinely moved among cages where they come in contact with new cagemates. Bedding and other debris can fall from the cages in one row into uncovered cages in the rows below, or may be transferred from one cage to the next on the gloves of animal husbandry technicians. Thus rodents housed in barrier rooms in uncovered cages can be exposed to infectious agents either via direct animal-to-animal contact (the most efficient means of transmitting infection) or indirectly through aerosols, fomites, and personnel (Shek et al. 2005). Sentinels for Research and Mutant Rodent Colonies Research and mutant colony (i.e., principal) rodents are usually not available for HM because they are on study or needed as breeders for colony expansion, especially if they are of a GEM strain that suffers from low fertility. Furthermore, the principals may be immunodeficient and therefore inappropriate for serosurveillance. These situations call for the monitoring of separate sentinel animals; the diagnostic methodology determines the optimal type of sentinel. Outbred stocks, besides being comparatively inexpensive, are sensitive serology sentinels because they are susceptible to viral infections but resistant to disease (as manifested by early, robust seroconversion). Certain immunocompetent inbred strains may be inappropriate serology sentinels if they are resistant to infection with common adventitious agents, as illustrated by C57BL/6 mice, which are resistant to MPV infection (Besselsen et al. 2000), or if they are likely to succumb to lethal infection before seroconverting, as has been demonstrated for DBA/2 mice infected with Sendai virus (Brownstein et al. 1981; Parker et al. 1978). It is important to replace serology sentinels on a regular basis as research has found age-dependent resistance to infection for certain common rodent viruses, including MPV (Besselsen et al. 2000) and mouse rotavirus (Riepenhoff- Talty et al. 1985). Immunodeficient (and diseasesusceptible inbred) sentinels can enhance the diagnostic sensitivity of HM methodologies (e.g., gross and microscopic examination, microbiologic culture, and PCR) that depend on demonstrating the presence of pathologic changes or the etiologic agent. This is because infections are more likely to be pathogenic and persistent in immunodeficient animals than in immunocompetent hosts (Besselsen et al. 2007; Clarke and Perdue 2004; Clifford et al. 1995; Compton et al. 2004a; Ward et al. 1996). Sentinel Exposure Contact with the principal animals is the most efficient and reliable way to transmit infection to sentinels, so the use of contact sentinels should be routine in the critical microbio- Volume 49, Number

5 logic assessment of imported animals in quarantine. However, sentinels themselves can be sources of genetic and microbial contaminations (Pullium et al. 2004). It is possible to prevent genetic contamination either by removing female sentinels after 2 weeks of contact or by using castrated males. Furthermore, the use of sentinels from gnotobiotic (or limited-flora) isolator-maintained colonies substantially diminishes the risk of microbial contamination; surplus euthymic heterozygotes from isolator-reared nude mouse colonies can serve well for this purpose. The use of contact sentinels for routine surveillance is often not feasible, however, because it conflicts with the study protocol or the investigator considers it an unacceptable risk. Additionally, for the monitoring of microisolation cages, contact sentinels would have to be placed in or moved among many cages, which is risky and logistically complicated. Sentinels are therefore usually kept in separate cages on soiled bedding transferred from the colony cages. Isolators, with uncovered cages, facilitate the transmission of infection via aerosols and fomites with the placement of sentinel cages on the bottom row of the isolator cage rack. Exclusive reliance on soiled bedding to transfer infections to microisolation cage sentinels for routine health surveillance has proven to be problematic for a number of reasons. Certain enveloped viruses and host-adapted bacterial pathogens may not be transmitted efficiently or at all in this way (Artwohl et al. 1994; Compton et al. 2004b; Cundiff et al. 1995; Dillehay et al. 1990; Ike et al. 2007; Thigpen et al. 1989). Aerosol transmission of enveloped respiratory viruses to sentinels, however, has been effective in VCS by housing sentinels in cages with unfiltered exhaust air from colony cages (Compton et al. 2004b). In barrier rooms and isolators, because agents spread freely among uncovered cages, a high percentage of animals become infected. By contrast, the percentage of animals that become infected in colonies in microisolation cages can be quite low and the fraction shedding an agent still lower. Thus, even for agents readily transmitted in soiled bedding such as the rodent parvoviruses, MNV, mouse hepatitis virus (MHV 1 ), and the enterohepatic helicobacters (Compton et al. 2004b; Livingston et al. 1998; Perdue et al. 2007; Smith et al. 2007; Thigpen et al. 1989; Whary et al. 2000) the quantity of an adventitious agent in bedding pooled from many cages can easily be diluted below a dose infective to the sentinels. Sample Size Since its publication in 1976, the ILAR formula for binomial distribution has commonly been referenced as a way to estimate HM sample size (ILAR 1976). The formula provides accurate sample size estimates when applied to animal populations of 100 or more in which the transmission of infection is unimpeded (e.g., large colonies of rodents housed in uncovered cages). Although not suitable for determining sample size when monitoring soiled bedding sentinels housed in microisolation cages, the binomial distribution formula and others like it (Dubin and Zietz 1991) illustrate a number of generally applicable concepts (Clifford 2001). First, in contrast to what seems intuitive, sample size does not increase as the number of animals in the population goes up; instead, it is determined by the expected prevalence of infected or assay-positive animals. Second, even if an assay for an agent is 100% accurate, negative results for all animals tested do not prove that the population is free of the agent; instead, they provide a level of confidence that the prevalence of assay-positive animals in the population is below the assumed minimum. Finally, as the prevalence of positive animals decreases, the sample size required to achieve that same level of confidence increases. For example, assuming a minimum prevalence of 35%, which is the typical assumption for a barrier population, it is necessary to test at least seven samples in order to be 95% confident of finding a positive, according to the binomial distribution formula. When the prevalence of assay-positive animals is assumed to be at least 10%, the sample size needed to obtain a positive result with 95% confidence jumps to 29. Frequency of Sampling The frequency of sampling is driven by the historical rate of contaminations with extraneous agents that compromise the microbiological status of a colony (Selwyn and Shek 1994), which as mentioned is affected by the housing system. For instance, gnotobiotic and immunodeficient colonies are usually maintained in isolators (or microisolation cages) to achieve the high level of biosecurity necessary to sustain a defined or limited microflora from which opportunists are excluded. Since contamination of isolator-maintained gnotobiotic and limited-flora colonies with extraneous bacteria and fungi (as a result of physical defects in an isolator or inadequately disinfected supplies) is much more common than are adventitious viral infections and parasite infestations, bacteriology (on isolator swabs) is performed much more frequently than serology and parasitology. Conversely, for surveillance of commercial barrier rooms where viruses are the most frequent cause of adventitious infections and where contaminations with pathogenic bacteria and parasites are extremely rare, serology is performed more often than bacteriology and parasitology. Repeat Testing to Corroborate Positive Results for Sentinels in Microisolation Cages When the percentage of infected microisolation cages is low (as is often the case for reasons discussed below) or when sentinels are being tested for an agent not readily transmitted by soiled bedding transfer, the prevalence of assaypositive sentinels is also likely to be low. This complicates 320 ILAR Journal

6 the ability to determine the true microbiologic status of a colony because as the prevalence of assay-positive samples decreases, the fraction of positive results that are correct (the positive predictive value) also declines (La Regina et al. 1992; Zweig and Robertson 1987). It is therefore prudent to substantiate low-prevalence positive findings by retesting the same and additional animals by multiple and complementary diagnostic methodologies. The approach to repeat testing depends on the pathobiology of the etiologic agent and the available diagnostic methods. For example, MPV, MNV, and MHV are among the most common contaminants of SPF mouse colonies; they are enterotropic and thus are shed in feces and can be detected in mesenteric lymph nodes. Because immunocompetent mice shed MNV at high levels indefinitely, the use of PCR on fecal specimens from seropositive sentinels or their cagemates can reliably corroborate MNV seroconversion (Hsu et al. 2006). By contrast, sentinel mesenteric lymph nodes are preferable to feces for PCR to confirm positive MHV or MPV serologic findings, because in seroconverted mice viral genomic sequences persist in the mesenteric lymph nodes (Besselsen et al. 2000, 2002; Homberger et al. 1991; Jacoby et al. 1995), whereas viral shedding in the feces stops (Besselsen et al. 2007; Compton et al. 2004a). After PCR corroboration of positive sentinel serology, the next step is to verify infection of the colony and determine the number and location of microisolation cages containing infected animals. A high percentage of cages need to be sampled to have a good chance of detecting an infection localized to a few. If there are a large number of cages, the preparation and testing of many serum samples can be labor intensive and expensive. Alternatively, PCR can be performed on VCS exhaust filters or on fecal pools or swabs of cages to determine the location of animals that are shedding virus (Compton et al. 2004b; Henderson et al. 1998). The interpretation of PCR results requires a measure of caution, as detection of a microbial genomic sequence does not necessarily indicate the presence of infectious microorganisms, although a quantitative PCR can help distinguish low-level environmental contamination from an active infection. In this regard, proof of seroconversion is important evidence that an infection has occurred. Biosecurity Challenges from the Expanding Use of Genetically Engineered Mutant Rodent Models Where once researchers had to wait patiently for mutant strains of interest to arise by accident, now it is possible to make GEM rodent models in the laboratory, essentially at will, for investigating a variety of issues and disease conditions. Consequently, myriad GEM models have been generated at governmental, academic, and commercial institutions, with new ones added daily (Mobraaten and Sharp 1999). Because there are so many GEM rodent models and the demand for most is small, commercial production is for the most part not economically viable. Colonies to supply GEM rodent models are therefore maintained at many research institutions, where biosecurity, husbandry, and HM practices are quite variable. For example, research institutions often keep both SPF and non-spf populations on the same site and frequently import new models, thereby increasing SPF rodents risk of exposure to adventitious agents from escaped or transferred rodents, personnel acting as mechanical vectors, and fomites such as inadequately disinfected supplies and shared equipment. As a result, parasite infestations and microbial infections, largely eliminated by commercial SPF rodent suppliers, have once again become prevalent (Jacoby and Lindsey 1998) and the frequent exchange of GEM models among institutions poses considerable challenges to the SPF status of research colonies. In recent years, the reliability with which major SPF rodent vendors have been able to exclude and monitor for adventitious infections has largely obviated the need to quarantine and perform HM on vendor animals before releasing them for use in studies. Quarantine of vendorsupplied rodents may still be appropriate, however, if the rodents have not been shipped on dedicated, disinfected transportation, especially when the animals will be distributed to many rooms as sentinels. On the other hand, quarantine of GEM rodents from noncommercial sources is normally necessary for the reasons discussed. This is true even when importing rodents that are presumably SPF according to HM results as these results may not accurately represent the source colony s current health status, because of limitations in the diagnostic methods, infrequent monitoring, or testing of too few and inappropriate specimens. In addition, imported animals might become contaminated while in transit. These concerns notwithstanding, in a retrospective study of a riskbased import and quarantine program (Otto and Tolwani 2002), the vast majority of HM reports from source institutions indicating that shipments of imported mice were SPF were confirmed by negative test results during quarantine. This study indicated that HM reports for source colonies are suitable for assigning animals for import to contamination risk categories, which serve as the basis for approval or rejection of importation and for the priorities and conditions of the quarantine. Examination and analysis of HM reports can also determine whether to quarantine high-risk rodent imports (i.e., those from colonies of unknown status or contaminated with one or more agents on the importing institution s SPF exclusion list) separately from presumably SPF imports. Such separation reduces the risk of contamination of SPF imports in quarantine and improves the capacity to accommodate requests for low-risk imports, which is particularly important given the increasing exchange of mutant strains among institutions. While in quarantine, high-risk animals in particular should be housed in isolation units in a manner that not only contains the agents with which they are infected but also Volume 49, Number

7 protects them from further microbial contamination. Containment practices can include keeping quarantine room air pressure negative to the pressure in common corridors, providing HEPA filtration of isolation unit exhaust air, opening microisolation cages and handling animals in a class II biological safety cabinet (instead of a horizontal laminar flow hood), placing materials for disposal in sealed containers or disinfecting them before their removal from the quarantine room, and rigorously controlling personnel access. Rederivation by embryo transfer (Van Keuren and Saunders 2004) or hysterectomy is the most reliable method of eliminating adventitious agents, with transfer of neonates to SPF foster dams as an effective, perhaps less costly alternative that preserves valuable breeders (Huerkamp et al. 2005; Lipman et al. 1987; Truett et al. 2000; Watson et al. 2005). Treatment with anthelminthics (Boivin et al. 1996; Huerkamp et al. 2000) and antibiotics (Foltz et al. 1996; Goelz et al. 1996) can cure animals of certain parasite infestations and bacterial infections, respectively; however, it is important to bear in mind (1) the potential for these treatments to be less safe or efficacious in GEM strains than in other rodents and (2) the types and amount of testing needed to verify efficacy with a high level of confidence. Conclusion Few would dispute that the major challenge to maintaining the SPF health status of research colonies is the dramatically expanding production, use, and importation of GEM strains at many institutions. This challenge and the continued discovery of indigenous rodent agents, which are then found to be common contaminants of presumably SPF colonies, have reemphasized the importance both of rigorous biosecurity and of routine HM. As reviewed, key biosecurity measures include rederivation and the housing of rodent colonies in room- to cage-level barrier systems to eliminate and exclude (and/or contain) microbial contaminants, respectively. Microisolation caging systems have become popular for housing GEM and other SPF rodent colonies, especially in hightraffic research facilities, because they are versatile and provide a highly effective cage-level barrier to the entry and spread of adventitious agents. HM is needed to verify the effectiveness of biosecurity measures as well as to determine whether to proceed with an importation (or transfer) and to release imported rodents from quarantine. Thus, accurate HM results are crucial for mitigating the risk that microbial contaminants will be introduced and spread in exchanges of mutant rodent strains among investigators and research institutions. Sampling choices (e.g., size, type, and frequency) have a substantial impact on how accurately HM results correspond to a colony s health status. False negative findings due to insufficient sample size or inadequately exposed sentinels are more likely when monitoring colonies housed in microisolation cages versus uncovered cages. This is because microbial contaminants can be freely transferred among uncovered cages via aerosols, fomites, and people, whereas microisolation caging systems inhibit these indirect modes of transmission. The percentage of infected animals is therefore often lower for colonies housed in microisolation cages than for those in uncovered cages. As the prevalence of infection decreases, it is also less likely that pooled soiled bedding will contain a sufficient amount of an agent to infect and elicit seroconversion in sentinels. To reduce the probability of missing low-prevalence adventitious infections of colonies housed in microisolation cages, it is important to consider approaches for increasing sentinel exposure as well as other monitoring methodologies to augment sentinel testing. For example, it is possible to improve the efficiency with which adventitious agents are transmitted to sentinels in soiled bedding by transferring bedding more frequently and by reducing the number of colony cages per sentinel cage, although these options also add to the cost and labor of HM. Interestingly, one study found that the soiled bedding transfer of MPV and MHV to sentinels was more efficient for mice housed in VCS than for those in static microisolation cages (Smith et al. 2007). The study authors surmised that this was the result of virus being inactivated by higher concentrations of NH 3 in static microisolation cages or being better dispersed in VCS. Regardless of the mechanism, this study indicates that the effects of factors such as ventilation and bedding type on the efficiency of soiled bedding transmission of infectious agents (particularly those that are less environmentally stable) warrant further investigation. Soiled bedding sentinel exposure can be augmented by other modes of transmission, including placing sentinels in contact with colony animals (although the routine use of contact sentinels for monitoring microisolation-caged colonies is largely impractical for reasons discussed) or housing VCS sentinels in cages supplied with unfiltered exhaust air from colony cages. The main alternative to sentinel testing for detection of microbial contaminants is to perform PCR assays on colony animal and environmental specimens where the concentration of prevalent adventitious agents is expected to be high. Such specimens include feces or fecal pools (since most prevalent agents are enterotropic) and room, rack, or cage exhaust filters, with high concentrations of potentially agent-laden particles. PCR results might be positive without infectious microorganisms being present or false negative because the agent assayed is not environmentally stable or is no longer being shed in amounts sufficient to detect in the tiny quantity of sample tested. The use of microbial PCR testing of environmental and colony specimens is therefore advisable as an adjunct to, and not a replacement for, sentinel monitoring. 322 ILAR Journal

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