Special Topic Overview. Rodent Quarantine Programs: Purpose, Principles, and Practice

Transcription

1 Laboratory Animal Science Copyright 1998 by the American Association for Laboratory Animal Science Vol 48, No 5 October 1998 Special Topic Overview Rodent Quarantine Programs: Purpose, Principles, and Practice Jerold E. Rehg and Linda A. Toth Abstract In animal research, validity and reproducibility of data are critically influenced by the microbial status of the experimental animals. One of the most crucial aspects of assuring quality in animal research is providing research personnel with confidence that experimental results will not be invalidated due to interference caused by infectious disease. An effective quarantine program is essential to providing this assurance. Quarantine programs are generally instituted to prevent the introduction of rodent pathogens into established specific-pathogen-free colonies in a facility. Therefore, programs should be designed to isolate newly acquired rodents until their health status can be determined and to maximize the probability that microorganisms of interest will be detected before the animals are introduced into (and thus, could potentially contaminate) established colonies. Important principles that are critical to designing an effective quarantine program will be discussed here, as will the practical implementation of these principles. Although quarantine programs may be costly in terms of time and effort, these costs must be balanced against the potential costs of disease outbreaks that could invalidate long-term studies, alter normal biological baselines, and cause the loss or necessitate re-derivation of rare or valuable strains of rodents. Reducing the incidence of quarantine failures through appropriate program design and implementation helps to maintain the confidence of research personnel in the value of quarantine programs and in our competence as specialists in laboratory animal management and as partners in the research process. The value of any research project is fundamentally dependent on the validity and reproducibility of the data. In animal research, validity and reproducibility are critically influenced by at least three important variables: genetic background, physical environment, and microbial status. Absolute control of these variables would theoretically culminate in the attainment of identical biological responses from individual animals tested under equivalent experimental conditions. Therefore, the goal of quality-assurance programs in laboratory animal management is to minimize these variables. Animal health-assessment programs serve the important function of reducing research interference due to infective agents. To be maximally effective, the health of the animals must be assessed before and during their experimental use. Quarantine programs accomplish the former appraisal, whereas clinical assessment and sentinel monitoring programs accomplish the latter. Quarantine programs are generally instituted to prevent the introduction of rodent pathogens into established specific-pathogen-free (SPF) colonies in a facility. Therefore, programs should be designed to isolate newly acquired rodents until their health status can be determined and to maximize the probability that microorganisms of interest will be detected before the animals are introduced into (and Department of Infectious Diseases, Comparative Medicine Division, St. Jude Children s Research Hospital, Memphis, Tennessee thus could potentially contaminate) established colonies. Achieving this goal requires consideration of two factors: the likelihood of dispersion of microorganisms of interest throughout the quarantined population during the quarantine period, and the interval necessary for the exposed rodents to undergo seroconversion or for the microorganisms to proliferate to detectable numbers. The first consideration requires development of a sampling strategy that will provide an acceptable probability of detection, and the second requires determination of an appropriate duration of quarantine. A rigorous, highly effective program may require prolonged quarantine periods and/or sampling and testing of large numbers of animals, thereby engendering cost to the institution and users as well as delays in the availability of animals for use in research. Because of these considerations, a cost-benefit analysis is a prerequisite to designing a rodent quarantine program. Institutions must balance the cost of a disease outbreak in terms of loss of animals and research time against the cost of maintaining animals in quarantine for a predetermined period and conducting the necessary surveillance procedures. Ideally, this analysis will result in determination of a risk factor that is acceptable and defined. For example, some institutions may determine that a high level of risk is acceptable on the basis of either the type of research done at the institution or the cost of maintaining a rigorous quarantine program, whereas others may require a high degree of assurance that 438

2 Special Topic Overview newly acquired animals will not threaten ongoing research or valuable colonies. Principles of an Effective Rodent Quarantine Program A fundamental concept essential to designing an effective quarantine program is the understanding that providing an acclimation period for newly acquired rodents is not equivalent to maintaining an effective quarantine program. Although adequate animal acclimation is crucial to obtaining valid experimental data, and rodents can undergo acclimation during the quarantine period, an effective quarantine program requires evaluation of newly acquired animals to determine that their health status meets predefined criteria. Intervals required for acclimation of rodents to new environmental conditions can differ from those required for quarantine (1 5). The two basic types of quarantine programs are passive quarantine and active quarantine (6). In passive quarantine programs, newly acquired animals are housed in an isolated location and are not manipulated experimentally for a predetermined period. During this period, they are clinically evaluated for signs of illness. The passive approach to quarantine is based on the assumption that infected animals will either develop clinical signs of disease or recover from subclinical illnesses and stop shedding the causative organism during the quarantine interval. The main problem inherent with this approach is that many of the common rodent infective agents that adversely affect research cause subclinical disease that may be associated with prolonged or intermittent shedding of the infective organism (7). Latently and subclinically infected animals pose a serious potential threat to the health of established colonies because, after release from quarantine, such animals may shed microbes if they become immune suppressed or stressed, perhaps from experimental use. They therefore may be a source of infection for other populations in the facility, potentially causing susceptible strains to have poor reproductive performance, overt clinical illness, or death. Moreover, even subclinical infections can alter biological responses (7, 8), thereby possibly invalidating experimental conclusions by inducing changes other than those caused by the experimental treatment (9). Because of the assumptions inherent in the use of passive quarantine systems, their durations should be longer than those of active quarantine programs. Passive quarantine is not recommended for institutions requiring a high degree of animal-health assurance because of the possibilities of undetected subclinical disease and prolonged microbial shedding in animals released from quarantine. In active quarantine programs, animals are kept in an isolated location for a predetermined period. During or after this interval, either the quarantined population itself or SPF sentinel animals housed in the same or adjacent cages are tested for various microbial contaminants, using techniques such as bacterial culture, serologic testing, fecal examination, and histologic examination. Several considerations are important in designing an effective active Table 1. Some considerations in designing a quarantine program Confidence level of detection Sample size Duration of quarantine Housing conditions False negatives Risk of infection Source of animals Shipment method Cost factors Monetary cost Research time Scientific considerations Value of established colonies Nature of the research quarantine program (Table 1). Most critical to the effectiveness of the program are several interacting principles that determine the level of statistical confidence that target microorganisms will be detected in newly acquired animals. The first principle involves determination of the number of animals to be sampled. Adequate numbers must be tested to ensure an acceptable probability that an adventitious organism of interest would be detected if it were present in the quarantined cohort. For our purposes, a cohort can be defined as a group of animals introduced simultaneously into the same secondary enclosure in quarantine (e.g., one shipment housed in one isolator, cubicle, or room). If each animal in the cohort will not be tested individually, one should apply principles of probability statistics to determine how many animals must be sampled to reveal an infection that is potentially present in an unknown (and perhaps low) proportion of the population. Theoretically, sufficient animals should be tested to provide an acceptable level of confidence that the target organism with the lowest expected incidence would be detected. Agents with low prevalence rates are more difficult to detect on a statistical basis; therefore, to achieve statistical confidence of detection, greater numbers of animals should be tested. Estimates of the prevalence of important pathogens in various rodent populations have been published (7, 10), but from a practical perspective, actual prevalence rates can change markedly and rapidly with time, particularly within specific colonies. Estimation of expected prevalence requires consideration of many factors, including general prevalence and transmissibility of the target agent in rodent populations, source of the animals, reported health status of the animals, and method of shipment. In our opinion, prevalence rates must ultimately be estimated largely on the basis of professional judgement. Charts and formulas are available for estimating the minimal sample size necessary to detect infections in various percentages of the test population to a specific level of confidence (7, 8, 11, 12). To increase confidence levels, greater numbers of animals must be tested. For example, random sampling and testing of 10 or 11 animals per cohort will yield 89 to 90% confidence that an infection will be detected, assuming a minimal 20% infection rate and a total population 100; in comparison, 14 or 15 animals must be tested to achieve 95% confidence (7, 8, 11, 12). Fewer samples are necessary if the total population is smaller, but testing a minimum of 10 animals per cohort has been 439

3 Vol 48, No 5 Laboratory Animal Science October 1998 Table 2. Factors that potentially influence duration of a quarantine period Type of caging: open vs. microisolator Type of secondary enclosure: open room vs. cubicle vs. isolator Animal sampled Research animal vs. sentinel animal Age, strain variables Sentinel exposure method Direct contact Aerosol Bedding transfer Target organism(s) Transmissibility Method of detection: serology vs. culture vs. other Table 3. Detection of antibody against mouse hepatitis virus in mice after exposure to an infected mouse Weeks Serologic response (% of animals tested) after exposure Positive Negative Equivocal A mouse known to shed mouse hepatitis virus (MHV) long term was introduced into a cage in the top row of a 36-cage rack for a 24-h period (see text and Figure 1 for additional details). After removal of the index mouse, the remaining mice (three per cage) were evaluated weekly for antibodies against MHV, using an in-house ELISA system (Organon Technicon, Durham, N.C.). Similar proportions of seropositive mice were obtained from a rack in which the index cage was located in the bottom row of the rack, although the distribution of positive mice on the rack differed (see Figure 1). recommended as a practical standard (12). The second principle that is crucial to designing an effective quarantine program involves determination of an adequate duration of quarantine. Theoretically, the quarantine interval should permit sufficient time for exposure, infection, and seroconversion of a proportion of the cohort that is consistent with a predetermined target confidence level of detection and sampling scheme. Typically, these processes require at least 2 to 4 weeks, which represents the minimal time necessary for infected animals with an uncertain date of infection to generate a measurable antibody response. The practice of testing newly acquired animals as soon as they arrive may reveal an infection if at least some of the animals are already seropositive and if adequate numbers of animals are tested. However, if animals are infected in transit, perhaps none of them will be seropositive, although many may be incubating an infection. Testing these animals on arrival would not reveal the infection, and in fact, the animals would ideally have to be assessed again after an appropriate interval. In our opinion, this approach is not cost effective, because animals will usually have to be tested twice. The advantage of waiting before conducting the initial evaluation is that a larger proportion of animals will have detectable antibody, thereby increasing the likelihood of detecting the agent of interest. This approach is more cost effective because only one serologic assessment is required, rather than two. Another important concept relevant to duration of the quarantine is that the quarantine period begins whenever the most recently acquired animals are introduced into the same secondary enclosure (13). For example, if new animals are introduced into a given secondary enclosure of a quarantine facility on day 21 of a 28-day quarantine, a new Figure 1. Rack location of cages containing mouse hepatitis virus (MHV)-seropositive mice. The block diagrams represent two standard 36-cage mouse racks composed of six rows of cages (12 x 9 x 6 in.) with six cages in each row. Each cage housed three adult female CBA/CaJ mice in solid-bottom caging with automatic watering, wood chip bedding, and a standard open grill top without filter tops. Cages were changed weekly at the time of blood collection. Cage changing and blood collection began in the cage farthest from the index cage and progressed toward the index cage (i.e., for rack 1, blood collection began in the lower right cage and progressed from right to left on each row from bottom to top of the rack; for rack 2, it began in the upper right cage and progressed from right to left on each row from top to bottom of the rack). The technician performing cage changing and blood collection handled mice using gloves that were dipped in a chlorine dioxide solution between animals or forceps that were sanitized by immersion in chlorine dioxide solution between cages. Each rack was located in a 4 x 6-ft cubicle. Non-recirculated air was supplied from the ceiling at a rate of 25 air changes/h and was exhausted near floor level. Cage changing and blood collection took place within the closed cubicle. A mouse known to shed MHV long term was introduced into either the top left or bottom left cage (indicated by double border) of each rack for a 24-h period. Numbers in the blocks on the diagrams indicate the number of mice in that cage that were seropositive for MHV 3 weeks after removal of the index mouse. Absence of a number indicates that all three mice in the cage tested seronegative for MHV. Both racks had 19 to 20% incidence of seropositive mice. 28-day quarantine period must be initiated for all animals in that area. This precaution is essential to ensure that older animals do not acquire infection from the new acquisitions, yet fail to undergo seroconversion in detectable numbers prior to prerelease testing. A number of variables are relevant to selecting an appropriate duration for quarantine (Table 2), but little empiric information concerning minimal durations is available. Suggested durations for quarantine programs vary from 1 to 6 weeks, with more extensive testing recommended for animals that will be added to breeding colonies (13 16). In one study, seroconversion of sentinel mice introduced into rooms containing infected experimental rodents that were housed under a variety of conditions was evaluated. This study indicated that periods ranging from 2 to 25 weeks could be required to detect seroconversion of sentinels, depending on the infective agent and the personnel traffic through the room (17). We evaluated the spread of mouse hepatitis virus (MHV) in a simulated quarantine environment (Table 3, Figure 1). To model shortterm contact with an infected mouse (as might occur during transit), we introduced one mouse previously documented to chronically shed MHV into an index cage of a 36-cage rack located in a cubicle (Figure 1). The infected mouse remained on the rack for a 24-h period and was then removed. In separate studies, the index cage was located on either the top or the bottom row of the rack. Weekly 440

4 Special Topic Overview serologic evaluation of the cohort indicated that a period of 3 weeks was required to achieve the 20% incidence of infection that is considered adequate for a sample size of 10 to 11 and an 89 to 90% confidence level; 4 weeks were required for most animals to undergo seroconversion (Table 3). Statistical modeling on the basis of these data confirmed that evaluation of 10 randomly selected mice would detect infected animals in 90% of the test cases. On the basis of these data, we adopted a 3-week minimal quarantine duration for animals entering the St. Jude Children s Research Hospital (SJCRH) Animal Resource Center. However, longer intervals might be required if animals were housed in a larger secondary enclosure. Intervals of fewer than 2 weeks do not allow sufficient time for the dual requirements of spread of infection throughout the quarantined population and generation of specific antibodies in exposed animals, and should therefore be considered an acclimation period rather than an effective active quarantine period. If the infection rate in a quarantined cohort is low, microbial contamination may not be detected unless an isolated contaminated cage is sampled by chance. Therefore, the third principle of designing an effective quarantine program is the establishment of conditions that facilitate detection of adventitious microorganisms. Housing systems that permit the spread of adventitious microorganisms between cages increase the likelihood that a high proportion of the quarantined population will undergo exposure and seroconversion during the quarantine period. Such housing systems should increase the proportion of infected animals in the population and, thereby, increase the confidence level for detection at low sample sizes. Unless each cage of animals will be sampled, microisolation caging is generally contraindicated for quarantine of immune-competent animals. Microisolator caging restricts infection to limited groups of animals and retards exposure of other animals within the cohort (18, 19), thereby reducing the likelihood of obtaining a level of infection that will be detected in a random sample of animals. Air-flow patterns within the quarantine room or cubicle can influence the pattern of dispersion of microbial contaminants, and consideration of air-flow directions within quarantine rooms or cubicles can potentially suggest sampling strategies that would reduce the numbers of tests needed to reach a target confidence level of disease detection. In our study of MHV seroconversion in a simulated quarantine environment, the rack housing the experimental cohort was located in a cubicle in which air entered from the ceiling and was exhausted near floor level. Serologic data collected 3 weeks after exposure indicated that mice housed on the bottom row of the rack were most likely to undergo seroconversion (Figure 1). Statistical analysis on the basis of this finding indicated that evaluation of 10 mice selected from the bottom row would provide 99% confidence of identifying an infection, whereas random sampling of 10 animals would provide only a 90% confidence level. This analysis suggests that sampling mice from cages located near exhaust ducts might permit detection of microbial contamination within the enclosure using lower numbers Table 4. Detection of MHV seropositive sera by reference laboratories ELISA results IFA results Sample In-house Reference laboratory In-house Reference laboratory no. A B C D A B C D /- - +/- + ND +/- + +/- 2 + _ +/ ND +/ _ ND +/ / Four mice that were seropositive for MHV during quarantine screening were anesthetized and exsanguinated. Serum prepared from each animal was aliquoted and frozen. Samples were tested in house, using the ELISA and an indirect immunofluorescent assay, and were submitted to four independent reference laboratories. ND = not determined by laboratory due to negative ELISA results. of samples. However, use of targeted rather than random sampling of the cohort would be strongly dependent on the assumption that the targeted cages had a higher incidence of infection than did other cages on the rack. The final principle relevant to the design of quarantine programs is consideration of the sensitivity and specificity of the testing procedures used for animal evaluation. Many programs rely on an enzyme-linked immunosorbent assay (ELISA) for preliminary serologic evaluation and confirm positive findings by use of an indirect immunofluorescence assay (IFA). These tests can be performed either in house or by reference laboratories. In our quarantine program at SJCRH, we routinely perform the ELISA and IFA in house to expedite test completion and release of animals from quarantine. If test-positive sera are detected, another blood sample is collected from each seropositive animal and from any previously untested cagemates; sera are then submitted to a reference laboratory for confirmation. While using this strategy, we have noticed that the results reported by various reference laboratories evaluating identical aliquots of serum often vary diametrically (Table 4). Others report similar discrepancies (20). Such observations suggest that unidentified samples known to be seropositive and seronegative should be submitted with clinical samples as a means of monitoring the accuracy of reference laboratories. Reports of positive and negative results from individual reference laboratories should be evaluated critically and perhaps confirmed elsewhere, particularly if rare or valuable animals are involved. Although our example focused on serologic testing, sensitivity and specificity are also important considerations for other types of diagnostic tests (e.g., parasitologic and microbiological testing). Quarantine Programs in Practice A variety of issues influence the design and implementation of quarantine programs in individual research institutions. Some authorities state that every animal entering a facility, regardless of source, should undergo quarantine (9, 13, 14). However, a key issue relevant to establishing a practical program involves determining the conditions under which quarantine is deemed necessary or cost effective. These conditions can vary depending on the needs of individual research institutions. Relevant variables include the source of newly acquired animals, the method by which animals are shipped to the recipient institution, the value of established colonies, and even the nature of the 441

5 Vol 48, No 5 Laboratory Animal Science October 1998 Table 5. Summary of 1996 quarantine survey Type of rodent quarantine program No. of institutions No quarantine regardless of source 6 (12%) Quarantine only rodents from non-approved sources 12 (57%) Quarantine rodents from approved sources if shipped by air 3 (6%) Quarantine all rodents regardless of source 12 (24%) Duration of quarantine No. of institutions Rodents from approved sources Any means of shipment 1 week 8 2 weeks 2 3 weeks 2 Shipment by air 3 weeks 1 4 weeks 2 Rodents from nonapproved sources 1 week 7 2 weeks 2 3 weeks 6 4 weeks 7 >4 weeks 6 Of the 100 institutions surveyed by mail, 49 responded (42 academic institutions and 7 pharmaceutical companies). At some institutions, quarantine durations differed for rodents from approved versus nonapproved sources. Quarantine durations provided by those institutions are included in all appropriate categories. research conducted at the institution (Table 1). For example, although many types of research can be adversely affected by clinical or subclinical disease processes, institutions with large programs in disciplines such as immunology may be seriously compromised if adventitious microorganisms infect standing experimental colonies. Subclinical infectious conditions could cause alterations in baseline values, increased variance across animals, or lack of reproducibility (21), and would be particularly costly in terms of research time if long-term studies were affected. Moreover, a potential complication for short- and long-term studies in most, if not all, scientific disciplines is that experiments may be completed and the findings reported without the knowledge that infectious processes had influenced the acquired data. If identified retrospectively, this situation could necessitate retraction and/or re-interpretation of findings (22, 23). If subclinical infections were present but never recognized, distorted research data could be collected and published (9). In 1996, we conducted an informal mail-in survey of 100 institutions from which we elicited information regarding quarantine policies. Results of this survey indicate that quarantine programs vary markedly in terms of duration of quarantine and types of rodent shipments included in the program (Table 5). Notably, many institutions maintain less stringent quarantine requirements for animals from approved vendors than for rodents from nonapproved sources. Arguably, the rigorous health-assurance programs maintained by reputable vendors may justify a less rigorous program for their animals. However, even established vendors can experience animal-health problems. For example, in 1992, our quarantine program at SJCRH detected a vendor outbreak of rat sialodacryoadenitis virus before the contaminated animals were released from quarantine, thereby allowing us to avoid contamination of our research colonies. Table 6 summarizes major aspects of the mouse quarantine program at the SJCRH Animal Resource Center, which requires that all newly acquired rodents, regardless of source, undergo a minimal 3-week quarantine prior to sampling and testing for common rodent pathogens. The practice of directly introducing newly acquired animals from known acceptable sources into rooms housing research animals requires the assumption that the new animals have not become contaminated during transit (15). Unfortunately, the potential for animal contamination during transit complicates even the status of high-quality animals (16). This risk can be reduced if animals are shipped from the vendor directly to the recipient institution via dedicated trucks that carry only SPF rodents. However, the risk of contamination is significant if rodents are shipped via air freight. Findings in our quarantine facility support this possibility. Since 1990, we have tested approximately 200 shipments of mice (bi-weekly shipments of 30 to 60 boxes for approximately 8 years). In three instances, we detected MHV in quarantined animals (December 1990, January 1993, and May 1996). These occurrences were not associated with vendor outbreaks, and all animals had been shipped by air freight. Storage of rodent-shipping containers in common holding areas for air freight can permit contact with and contamination by wild rodents or contaminated vendor rodents. The likelihood of contamination is undoubtedly increased if shipping containers are damaged during transport. Therefore, technicians responsible for receiving rodent shipments must be highly attentive to examining cartons for signs of damage or rodent contamination. The preceding data indicate that even animals obtained from reputable vendors can shed infective microorganisms while housed in quarantine areas. This possibility mandates use of operational procedures to reduce the likelihood of exposing established colonies to infected animals housed in quarantine. If possible, quarantine facilities should be physically isolated from the rest of the facility (13). If an isolated area is not available, negative-pressure flexible film isolators can be used (13). Equipment used in quarantine suites should be thoroughly sanitized before and after use (24). Prior sanitization helps to ensure that infective agents detected in quarantined animals were introduced from external sources and not from within the facility. Ideally, a dedicated pass-through autoclave should be incorporated into a well-designed quarantine suite. Airflow directions within quarantine suites should be carefully monitored to ensure that cubicles or rooms housing quarantined animals are maintained at negative pressure relative to common corridors. Thus, air should move from areas with less risk of contamination to those with greater risk. Animal-husbandry personnel can easily monitor and record the direction of air flow on a daily basis if small, flexible plastic strips are placed in doorway ventilation grills (Figure 2). Use of this simple technique or a similar method can complement sophisticated but remote mechanical monitoring systems. Ideally, dedicated personnel should be assigned to provide husbandry care to quarantined animals, thereby reducing the risk of inadvertent movement of personnel from potentially contaminated areas to areas housing established 442

6 Special Topic Overview SPF colonies. If dedicated personnel are not available, animals in quarantine should be serviced as the final task at the end of the work day (24). To reduce the likelihood of microbial spread between secondary enclosures within the quarantine suite, personnel should use a prescribed entry order in which the most recently acquired or the most highly suspect animals are serviced last. Because the health status of newly arrived animals is unknown during the quarantine period, manipulation of these animals should be avoided to the extent possible. The numbers of animals housed in each cage in quarantine should be minimized as much as possible to reduce the necessary frequency of cage changing. Reducing the frequency of cage changing limits the generation of aerosols that could mediate the spread of microbial contaminants beyond the quarantine facility and the amount of potentially contaminated caging that must be processed prior to establishment of the SPF status of quarantined animals. Immunosuppression has been suggested under some circumstances as a means of revealing subclinical infections in quarantined rodents (25, 26), but use of this strategy Figure 2. Plastic strip on door grill indicating direction of air flow. Room technicians can easily monitor air-flow directions if a thin, flexible plastic strip is placed near room or cubicle air-supply vents in the door. A small label indicates the intended air-flow direction. This simple system can supplement sophisticated yet remote mechanical monitoring systems. Schedule: Duration: Mouse entry: Husbandry: Sampling: Testing: Schedule: Duration: Animal use: Husbandry: Sampling: Testing: Table 6. Mouse quarantine program at St. Jude Children s Research Hospital Approved vendors We operate two quarantine cycles/mo, using two separate suites of cubicles. Mice are received only during the first and third weeks of each month. Mice that have cleared quarantine are released, and the quarantine suite is sanitized before new animals are introduced into a suite. 21 days, regardless of the method of shipment. Shipping containers are carefully examined on the receiving dock, and damaged containers are rejected. Intact, unstained containers are sprayed liberally with Alcide on the receiving dock and are then transferred into quarantine suite. Mice from different vendors are housed in different cubicles. All feed, water, and caging materials, including racks, are autoclaved prior to use. To permit time for acclimation to the automatic watering system, mice are housed with two water bottles, but automatic watering also is maintained. Immune-competent mice are housed in open caging, and immune-compromised mice are housed in microisolator caging (Micro-Isolator, Lab Products, Inc., Seaford, Del.). Sentinels are added to the latter cages, if deemed necessary for detection of viral seroconversion. Quarantine personnel observe mice daily through the cubicle doors, but enter cubicles only to replenish food or water, to correct equipment problems, or to investigate unusual numbers of deaths. A dedicated technician is assigned to the quarantine suite. This technician is required to wear a fresh work uniform each day. Anyone entering the quarantine suite must also wear a disposable gown, gloves, shoe covers, face mask, and hair cover. This clothing is changed if multiple cubicles must be entered and is discarded within the quarantine suite before exiting. Weekend and holiday checks are performed as the final task of the day by the supervisor on duty. After entering the quarantine suite, husbandry personnel are not permitted to enter any other animal housing area until the next day and are required to shower prior to that time. Research personnel may not enter the quarantine suite, and experimental treatments or breeding cannot be initiated during quarantine unless exceptional circumstances apply. Samples for serologic testing are collected on day 21 of quarantine. Blood samples are collected from randomly selected mice to obtain a minimum of 20 samples/cubicle and at least one sample from each strain of mice housed in the cubicle. If mice are housed in microisolation cages, samples are collected in a hood, and every cage is sampled. Mice are anesthetized for blood collection, and then are individually numbered by ear notch. An appropriate notation is made on the cage card. All mice, regardless of source, are routinely tested for MHV, Sendai virus, mycoplasmas, and pneumonia virus of mice, which are considered likely potential contaminants that could be contracted from exposure to wild mice during transit. Tests for additional viruses may be conducted, depending on the intended housing location for the mice inside the animal facility. Initial testing is performed in house. If positive samples are detected, additional samples are collected from the index animal and from all cage mates. These samples are tested again in house and are submitted to at least one reference laboratory for confirmation. Modifications for nonapproved vendors Mice obtained from nonapproved vendors are housed in negative-pressure flexible film isolators during quarantine. Isolators are sterilized with formaldehyde gas between groups of animals. Minimal duration of 28 days ranging up to 56 days, depending on the source and suspected health status of the mice. Experimental use is not permitted, although in some instances breeding may be initiated after 14 days. Mice are housed in open caging in the isolator. Sentinel mice are introduced into the isolator if the quarantined animals are immune incompetent, or if they are too rare or too fragile to permit anesthetization for blood collection. If possible, one sentinel is introduced into each cage of cohort mice. Otherwise, 6 to 12 sentinels are placed in the isolator in open caging, and soiled bedding is added to their cages twice a week. Throat and rectal cultures, anal and pelage tape impressions, and fresh feces are collected on the day of arrival for parasitologic and microbiologic assessment. This testing is repeated when blood samples are collected for serologic testing. After an appropriate interval, blood samples are collected from all sentinel animals or from a representative number (at least one per cage) of cohort mice. After the sentinel mice are declared free of rodent adventitious viruses, they are submitted to the diagnostic laboratory for thorough parasitologic evaluation. Sera for 16-agent serologic panels are routinely submitted to a reference laboratory for analysis. 443

7 Vol 48, No 5 Laboratory Animal Science October 1998 Figure 3. Room log located outside the quarantine suite. This log often satisfies investigators needs for information regarding the status of quarantined orders. The log identifies the specific animal orders undergoing quarantine and provides an anticipated quarantine release date. can alter the time interval required for seroconversion of the quarantined cohort and increase the risk to standing populations by promoting excess microbial shedding. Even nonmanipulative activities such as breeding can permit transfer of microbial contaminants to new populations of susceptible animals. Research personnel sometimes believe that quarantine programs unnecessarily delay the research process, and they are often eager to use newly acquired animals as soon as possible. However, a policy of institutional and peer support for a quarantine program encourages researchers to incorporate the time required for quarantine into their research planning and discourages attempts to use animals experimentally during quarantine. To satisfy the investigators need for information regarding the status of quarantined orders, we maintain a room log outside the door of the quarantine suite (Figure 3). This log identifies the specific animal orders undergoing quarantine and provides an anticipated quarantine release date. This information can also be provided via computer networks. In special cases requiring animal manipulation prior to release from quarantine (e.g., harvesting fetuses that die in utero from timed-pregnant mice), research personnel can arrange to use animals either in flexible film isolators or under biohazard level-3 conditions. In these cases, however, investigators must be reminded that the collected tissues may harbor adventitious viruses and be cautioned not to inject these materials into immune-naive mice in the facility without prior mouse antibody production (MAP) testing. Special considerations apply if the newly acquired animals cannot be sampled directly. This situation arises most commonly in three situations: the new acquisitions are either rare or fragile, so that use of anesthesia for blood collection would pose an unacceptable risk of death; the mice do not make antibodies and therefore are not suitable for serologic testing; or institutional policy does not permit testing of animals intended for experimental use. In such instances, sentinel animals are generally substituted for the cohort population. Ideally, sentinels should be provided by the vendor or the shipping institution, differentiated in some manner from the experimental population (e.g., by identification number or coat color), and shipped in the same primary enclosure as the animals intended for experimental use. In that way, sentinel mice experience the same opportunity for exposure to microbial infection during transit as do the experimental animals. However, the vendor or shipper is often unable to supply suitable immune-competent sentinel mice. In that case, sentinel mice can be introduced at the recipient institution. Sentinel mice should obviously be immune competent, free of infectious disease, free of detectable antibody to organisms of interest, and old enough to have lost maternal antibody and to generate a robust primary antibody response. Strain may also be a consideration in some circumstances due to the well-known differences in susceptibility to various rodent viruses that exist even among immunecompetent strains of mice. Exposure of sentinel mice can be accomplished by overnight placement of the mice in empty shipping containers, by cohabitation of sentinel and quarantined mice, or by aerosol exposure with or without bedding transfer in the same secondary enclosure. Choice of the sex of cohabitating sentinels can be problematic. Female mice are generally preferred as cohabitating sentinels because direct introduction of sentinel males into a quarantined female population could result in unwanted pregnancies that might delay subsequent use of the animals for experimentation or breeding, and direct introduction of male sentinels into a quarantined male population could result in fighting that might impair the health of quarantined animals. However, because introduction of a female into a group of males can also evoke fighting, choice of the optimal sex for cohabitating sentinels is largely determined on the basis of professional judgement and experience. The strategy of transferring soiled bedding from the cages of the quarantined cohort to those of sentinel animals is often used as a means of promoting exposure and seroconversion of sentinel animals. For this strategy to be effective, infected animals must shed the organism into the environment, the organism must remain stable in the environment for an indefinite period, and sufficient amounts of the agent must be present in the transferred bedding to actually infect the sentinel population. In one study (27), daily bedding transfer from rats experimentally infected with sialodacryoadenitis virus caused seroconversion of immune-naive rats. However, rodent husbandry schedules typically involve a maximum of two changes of caging per week, and transfer of dirty bedding can often be conveniently performed only at those times. Studies involving a maximum of two bedding transfers per week vary in their conclusions about the effectiveness of soiled bedding as a fomite for transfer of murine pathogens (28 32). After a 3- week period, mites and MHV were detected more frequently in mice exposed to dirty bedding than in animals housed in the same room but not directly exposed to dirty-bedding (28). In another study, transfer of bedding from Helicobacterinfected mice caused infection and seroconversion of senti- 444

8 Special Topic Overview nel mice within 2 to 4 weeks (29). Seroconversion to Sendai virus was detected in mice after 10 to 15 days of exposure to contaminated bedding, but the effect was variable and was dose and strain dependent, resulting in the conclusion that the dirty bedding sentinel system was not effective for detection of Sendai virus infection (30). Others report that bedding transfer was ineffective in revealing Sendai virus infection in mice (31) and cilia-associated respiratory bacillus infection in rats (32). In studies using experimentally infected index mice, seroconversion occurred consistently in immune-naive weanlings housed for 1 week in soiled caging that previously contained orphan parvovirus-infected weanlings (33), but was less consistent in immune-naive mice housed in soiled caging that previously contained mousepox-infected adult mice (34) or parvovirus-infected pups (33). Taken together, these studies suggest that transfer of soiled bedding may promote detection of some rodent pathogens in some instances but that it may not be sufficiently reliable to serve as the foundation of a rigorous quarantine program. Reliance on transferred bedding as the primary method of exposing sentinel animals requires use of a longer quarantine duration, especially if mice are housed in microisolation caging, because sentinel animals will presumably not be exposed until caging of the quarantined cohort is changed. Another issue relevant to designing a quarantine program involves selecting the panel of infective organisms that will be evaluated in the quarantined cohort. This decision is influenced by the source of the animals and by the method of shipment to the recipient institution. In the SJCRH program, we have different requirements for rodents from approved and nonapproved sources (Table 6). On the basis of our extensive experience with the health quality of animals obtained from specific vendors, we make the assumption that mice from approved sources are infection free when they are crated by the vendor. However, we maintain a concern that the animals will become contaminated in transit via contact with wild rodents. On the basis of our review of relevant literature, we concluded that MHV, Sendai virus, pneumonia virus of mice (PVM), and mycoplasmas might be readily transmitted by contact with wild rodents during transit, and we therefore routinely survey all shipments of mice, regardless of source, for these organisms. For similar reasons, we evaluate rats from approved vendors for sialodacryoadenitis virus, Sendai virus, Kilham rat virus, PVM, and Mycoplasma pulmonis. When we obtain rodents from nonapproved sources, we routinely request that they be shipped to our facility in germ-free drums, which we supply. Samples for microbiologic and parasitologic evaluation are collected at the time of animal receipt and when blood samples are taken for serologic analysis. Serologic panels for mice from nonapproved sources comprise 16 organisms. Our sampling strategies typically assume an infection incidence of 20% and a target confidence level of 99%. A particularly problematic situation arises with regard to detection of parasites (e.g., mites and pinworms) in quarantined animals. Because these organisms must be detected Table 7. Sources of infection for laboratory rodents Newly acquired rodents Vendor vs. nonvendor sources Infection in transit Shipping containers: type and integrity Transportation method: air freight vs. dedicated truck Research supplies, equipment, and personnel Contaminated research materials (e.g., tumor cell lines, sera, antibodies, tissue culture supernatants) Contaminated equipment (shared centrifuges, incubators, animal equipment) Outside animals (pets at home, farm animals) by direct examination of the feces or coat of quarantined animals, sporadic shedding and/or presence in low numbers can make them difficult to detect, thereby undermining confidence that reports of negative results are truly negative. The possibility of mite and pinworm infections is a particular concern when animals are obtained from other research institutions, but infestations of vendor colonies also have been detected with surprising frequency during recent months. Prophylaxis of suspect shipments by treatment with anthelmintics, such as fenbendazole (35) and ivermectin (36 41), and/or with insecticides, such as dichlorvos (41 43) and permethrin (44), may be indicated under some circumstances, but treatments that reduce the number of organisms without causing eradication exacerbate the difficulties inherent in detecting these organisms during the quarantine period. In addition, when considering use of prophylaxis, the potential for toxicity must be considered (45 50). A final consideration relevant to safeguarding SPF colonies from microbial contaminants is the potential for introducing adventitious organisms by routes other than new animal acquisitions (Table 7). A rodent quarantine program should, therefore, be supported by evaluation ( quarantine ) of murine biological products prior to their use in experimental animals. These substances can be an important source of infection for laboratory rodents, and testing of tumor lines for rodent viruses is a historically recommended practice (6, 15, 51). Notably, recent reports document contamination of commercially supplied mouse serum with ectromelia virus (52) and of transplanted tumors with MHV, Staphylococcus aureus, and Pseudomonas aeruginosa (53). Such reports validate the need for caution when murine biologicals are used. Pet rodents or other animals maintained at home by research or husbandry personnel also can serve as a source of infection for research colonies (54). Conclusions One of the most crucial aspects of ensuring quality in animal research is providing investigators with confidence that experimental results will not be invalidated due to interference caused by infectious disease. An effective quarantine program is essential to providing this assurance. Designing an effective program requires defining program goals in terms of a target confidence level of detection for organisms of interest, and determining the sample size, quarantine duration, and housing conditions necessary to achieve this target. Although quarantine programs may be costly in terms of time and effort, these costs must be bal- 445

9 Vol 48, No 5 Laboratory Animal Science October 1998 anced against the potential costs of disease outbreaks that could invalidate long-term studies, alter normal biological baselines, and cause the loss or necessitate re-derivation of rare or valuable strains of rodents. The expense of quarantine programs and the research delays they engender mandate that programs have clearly defined goals and are carefully designed to ensure that they are operationally and statistically adequate for accomplishing those goals. Because quarantine programs are based on statistical inferences, they are always subject to some probability of failure, but failures may be more frequent if programs are based on inaccurate assumptions. Reducing the incidence of quarantine failures through appropriate program design and implementation limits their economic impact and helps to maintain the confidence of research personnel in the value of quarantine programs and in our competence as specialists in laboratory animal management and as partners in the research process. Acknowledgements We thank Joe Emmons and Mike Straign for technical assistance, the Department of Biostatistics and Epidemiology for statistical consultation, and Drs. Amy L. B. Frazier, Jack Hessler, and Peggy Danneman for review of preliminary versions of this manuscript. This work was supported in part by NIH grant no. CA and by the American Lebanese Syrian Associated Charities (ALSAC). References 1. Dymsza, H. A., S. A. Miller, J. F. Maloney, et al Equilibration of the laboratory rat following exposure to shipping stresses. Lab. Anim. Care 13: Grant, L., P. Hopkinson, G. Jennings, et al Period of adjustment of rats used for experimental studies. 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