Download Role of Housing Modalities on Management and Surveillance

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
B
ecause 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
01887 or email [email protected].
316
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 (HEPA1) 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 (PCR1) 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)
ILAR Journal
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
(SPF1) 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
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2008
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 CO2 and NH3) in static
microisolation cages were significantly elevated compared
to room levels, ventilated caging systems (VCS1), 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
NH3 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
317
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 (GEM1) 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
318
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
(MPV1) 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 (MNV1) (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
ILAR Journal
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
(HM1), 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
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2008
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 (RiepenhoffTalty 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 microbio319
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 (MHV1), 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.
tinels 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.
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 sen320
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
ILAR Journal
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
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2008
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
321
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 corre322
spond 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 NH3 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.
ILAR Journal
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