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4 Maintenance of Rodents Requiring Isolation GENERAL CONSIDERATIONS Certain mutations (i.e., nude mice, rats, hamsters; scid mice; and all multiple mutant strains carrying the homozygous nude gene) and experimental manipulations (e.g., sublethal irradiation) result in an immunodeficiency that is so severe that exposure of these animals to agents of even relatively low pathogenicity results in severe illness and death. For example, several studies have shown that athymic mice exposed to mouse hepatitis and Sendai viruses have survival times of less than 3 weeks (Sebesteny and Hill, 1974; Ward et al., 19761. However, in animal facilities that exclude pathogens to which they are susceptible, athymic mice exhibit a life span nearly as long as that of their euthymic counterparts (ILAR, 19761. It is therefore essential that the housing provided for these animals creates a barrier between the sus- ceptible host and infectious organisms. The Guide for the Care and Use of Laboratory Animals (NRC, 1985) states: "The caging or housing system is one of the most important elements in the physical and social environment of research animals" (p. 111. How- ever, equally important in the maintenance of a barrier are proper husbandry procedures, personnel management, and equipment maintenance. To ensure the health of pathogen-free animals, programs should be designed to monitor the colony by using clinical, serologic, pathologic microbiologic, and par- asitologic techniques (Hsu et al., 1980; Fox et al., 1984; Small, 19841. Because the well-being of pathogen-free animals is so strongly influenced 148

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MAINTENANCE OF RODENTS REQUIRING ISOLATION 149 by the housing system, special design and environmental control are of utmost concern. These systems are described below. SPECIAL FACILITIES AND EQUIPMENT Plastic Cages with Filter Covers This system consists of transparent plastic cages covered with securely fitted filter caps or covers. All components can be sterilized separately by autoclaving. The committee recommends the use of transparent plastic cages to facilitate routine animal observation without the need to open the cage more frequently than is necessary for sanitation and experimentation. These units represent the simplest solution to housing pathogen-free rodents, but they require rigid discipline and operating procedures if they are to be used as a primary barrier against disease (Sedlacek et al., 19801. HEPA- Filtered Laminar-Air- Flow Systems These units are composed of modular chambers, hoods, and racks that place cages under a positive flow of filtered air, independent of the room ventilation system. They can either be used to house rodents or to hold rodents while cages are changed. Transportable units are commercially avail- able (Figure 4-11; filters for whole-room application usually must be custom designed. Typically, these devices use high-efficiency particulate air (HEPA) filters that are capable of removing 99.97 percent of air-borne contaminants 0.3 Em or larger. A plenum and blower fan in the unit distribute the ultra- filtered air evenly over the cages in a horizontal laminar fashion. When planning an animal room with laminar-air-flow devices. careful consideration must be given to the energy requirements for multiple units and the heat load that is added to the room by the operation of these units. Low-velocity vertical mass air flow may be a successful adjunct to horizontal laminar air flow; however, the effectiveness of vertical mass air flow by itself is controversial (Thigpen and Ross, 19831. Regardless of the manufacturer or type of HEPA- filtered air supply device used in a room housing immunodeficient rodents, there are three essential components of management: 1. careful inspection and scheduled changing of the prefilters; 2. routine monitoring of the air velocity gauge, which indicates air- flow rate through the HEPA filter (falling pressure requires corrective action); and 3. annual recertification of the HEPA filter unit by qualified service personnel to ensure that the HEPA filter is intact, properly seated, and not leaking.

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150 IMMUNODEFICIENT RODENTS A I R I N TA K E 1 1 1 1 1 1 1 1 PREFILTER_ ~ . BLOWERS _ PLENUM AIR HEPA FILTER ~ ~ llr() PERFO RATED LI ~ SC REEN ~ ~ ~ g ~1 it' . e LAMINAR FLOW CONFIGURATION it 110- STAIN LESS STEE L ADJUSTABLE SHELVES FIGURE 4-1 Diagram of a horizontal single-row laminar-air-flow cage rack for housing im- munodeficient rodents. Figure courtesy of Lab Products. Inc., Maywood NJ. Attempts should be made to minimize dust in rooms equipped with these units to reduce the potential for loaded filters and failing systems. Laminar-air-flow benches or cabinets, preferably on casters for easy man- agement, can be used as a work surface for cage changing and experimental manipulation of animals. The principle applied here is the same as that for housing units. The committee considers this technique to be a useful adjunct to proper husbandry procedures, especially in facilities where barriers are not available or animal use is less restricted. Germfree Isolators For complete exclusion of microbes, the ideal housing system is the pos- itive pressure isolator, such as those used to house germfree, or gnotobiotic, rodents. The most widely used isolators are made of flexible laminated vinyl plastic (ILAR, 1970, pp. 5-61. They can be chemically sterilized and easily adapted to specific needs. Isolators are also available in stainless steel, nylon,

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MAINTENANCE OF RODENTS REQUIRING ISOLATION 151 and polycarbonate (Plexiglass). All such units have a filtered air supply and exhaust. Food and supplies must be sterilized and passed into the unit through a sterile entry chamber. Although isolators are an ideal housing system for keeping immunodeficient rodents pathogen-free, they are labor intensive, limit the number of animals that can be cared for in a given space, and seriously impair the ability to manipulate the animals. Tn~livi~luallv Ventilated Cage Racks _ . ~ ,,, Ventilated racks that accommodate cages in enclosed cabinets or by sus- pension under closed tops and that supply air under positive or negative pressure or both are available. Depending on the intended use, these cage racks can supply HEPA-filtered air or animal room air. The efficacy of these systems in protecting immunodeficient animals from infectious agents has not been evaluated. SPECIALIZED HUSBANDRY General Considerations The predominant consideration in the husbandry of immunodeficient ro- dents requiring protection is the very low resistance of these animals to infections of all types. The degree of protection provided should be dictated by the goals of the intended research. Precautions essential to gerontological studies, for example, might not be necessary in studies lasting only 1 or 2 months. However, all infections, including inapparent ones, are a constant threat to successful experimentation. Inapparent infections can, in a sense, be even more harmful than overt infections because they might go undetected and their impact on the study might be overlooked. Care must be taken to ensure that the results of an experiment in which immunodeficient rodents are used reflect the parameters measured and not concomitant infection. Environmental Conditions General rules that should be followed to ensure efficient husbandry are presented below. Temperature Immunodeficient rodents have been satisfactorily maintained in rooms where the temperature is regulated between 23.3C (74F) and 27.8C (82F). It has been shown that the thermoneutral zone for nude mice is above that for haired mice (Weihe, 1984) and that thyroid function in nude mice is

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152 IMMUNODEFICIENT RODENTS impaired from birth (Pierpaoli and Besedovsky, 1975), resulting in hypo- thyroidism and poorly developed brown adipose tissue (Gripois et al., 1980~. Therefore, it has been suggested that maintaining nude mice at 30-32C (86.0-89.6F) and providing thyroid hormone substitution could improve their health and life expectancy (Weihe, 1984~. As a practical matter, these steps are rarely taken. Furthermore, excessively high room temperatures create husbandry problems, such as fermentation of feed and bedding, excess bacterial growth in watering systems, and an unpleasant work environment. Most managers find that a typical rodent room temperature of 21.1-23.3C (70-74F) plus filter caps, which the committee recommends universally for immunodeficient rodents, results in temperatures that are high enough for successful husbandry. Room temperature should be graphically monitored, and an alarm should be installed to signal a malfunction of the temperature control. Humidity, Ventilation, and Lighting Each of these factors has been specifically addressed in the Guide for the Care and Use of Laboratory Animals (NRC, 19851. Readers should refer to that document for details. Food and Water Food. Only sterilized or pasteurized diets should be fed because many commercial diets are contaminated with Enterobacteriaceae (Williams and Habermann, 19621. Sterilization of food reduces this potential source of infection. Vitamin-fortified, sterilizable diets are available and should be processed just prior to feeding. When diets are sterilized, the autoclave function must be carefully monitored by qualified personnel to ensure that sterilization has been achieved. It is also important that the vitamin content of the feed remains at recommended levels and that the diet is not excessively hard or clumped. Diets decontaminated by irradiation are also commercially available and can be considered as alternatives to steam-sterilized or pas- teurized diets. Water. Acidification of the drinking water with HCl to a pH of 2.5-2.8 has been found to be effective in controlling microbial contamination, in- cluding that with Pseudomonas spp. (McPherson, 19631. However, this pro- cedure may impair the action of antibiotics and vitamins placed in the drinking water. In addition, excess acidification and chlorination have been associated with abnormalities in macrophage and lymphocyte function (Fidler, 1977; Hermann et al., 19821. The acidification procedure is still commonly used,

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MAINTENANCE OF RODENTS REQUIRING ISOLATION 153 however, because it is a practical way to control microbial contamination (Reed and Jutila, 19721. Water bottles, stoppers, and drinking tubes should be washed and sterilized between uses. Bedding Bedding can also be a source of microbial contamination, and therefore, steam sterilization is recommended before use. Bedding material should not irritate the skin of rodents, especially those without hair. Ground corncob or fine hardwood chips are both acceptable, although the relatively poor absorbency of ground corncob bedding makes it less desirable. The addition of sterile, soft-tissue sheets to the cages of pregnant females for nesting material should be considered, especially for those strains that have a history of poor reproductive performance. CONTROL OF INFECTION Arrival of Stock from Commercial Sources and Transfer of Animals Movement of animals into a facility for immunodeficient rodents should be held to an absolute minimum unless the facility is dependent on outside supply sources. The single most likely cause of barrier failure is the uncon- trolled or ill-advised introduction of new animals into the barrier. In the case of barrier rooms operated as maintenance facilities for rodents from outside suppliers, the opportunities for failure exceed those of carefully managed, closed-colony facilities with integral breeding. In this case, it is essential that suppliers provide complete documentation of the health status of their colonies and that they be required to notify users immediately if they ex- perience a failure of the desired health status of their colonies. When it is necessary to introduce new animals to provide genetic stock for backcrossing or other procedures, prior planning is mandatory. Germfree, specific-path- ogen-free, defined-flora, or viral-antibody-free animals should be obtained if possible. On arrival, the containers must be examined for breaks and tears and the invoice must be examined to ensure that the animals are of the proper genetic and environmental history. Animals must be quarantined in a room separated from all other rodents until they are known to be free of disease. Rodents being transferred into a facility must be tested for pathogenic bacteria, intestinal protozoa, and murine viruses (Hsu et al., 1980; Fox et al., 1984; Small, 19841. Some pathogenic organisms, such as Pneumocystis carinii, can only be detected by histological examination of cohorts or off- spring of the stock to be introduced. In the case of athymic rodents, heter

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154 IMMUNODEFICIENT RODENTS ozygous littermates are ideal subjects for viral serology testing. If these are not available, gnotobiotic rodents or rodents from the barrier may be added to cages of prospective introductees to act as direct contact sentinels. These sentinels are examined serologically, 4-6 weeks later, for viral antibody titer. This procedure is sometimes referred to as cohabitation and is optimum when females are used as cohabitants to prevent fighting. Regardless of the methods used to establish the health status of the new stock, they should be as thorough as possible and conducted in a quarantine holding area operated as a carefully managed barrier. Rodents transferred out of the colony should not be returned. Within the barrier, animal injection, anesthesia, and surgical procedures should all be conducted under aseptic . . cone citrons. Disease Surveillance Immunodeficient rodents must be observed constantly so that sick animals are detected promptly. Sick animals should be removed immediately to a quarantine area and submitted to the laboratory for diagnostic studies. Di- agnostic tests to determine the cause of the illness should be conducted as soon as possible, and steps should be taken to prevent infection of the other animals. These tests should include complete necropsies, as well as bacte- riologic and viral antibody assays. Because immunodeficient rodents are not able to react immunologically against all viruses (Eaton et al., 1975), they occasionally present a false-negative report. Therefore, an accurate evaluation requires that both immunodeficient and immunocompetent rodents be tested for the viral status of the colony. Immunocompetent sentinel rodents should be distributed randomly in a room that houses immunodeficient rodents, and a single individual from each cage of sentinels should be examined period- ically for infectious diseases. A complete discussion of diseases that commonly infect rodents can be found in ILAR ~ 1974), Wagner and Manning ~ 1976), Baker et al. ~ 1979a), Foster et al. (1982), and Fox et al. (19841. Many of the infectious agents discussed in these documents produce far more severe manifestations of disease in immunodeficient rodents than they do in their normal counterparts.