Housing and Maintenance

CHAPTER

Housing and Maintenance

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Hans J Hedrich Institut for Versuchstierkunde, Medizinische Hochschule, Hannover, Germany

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Horst Mossmann Max-Planck-lnstitut for Immunbiologie, Freiburg i. Br.,Germany

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Werner Nicklas Zentrales Tierlabor, Deutsches Krebsforschungszentrum Heidelberg, Germany

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Introduction

Husbandry

Proper housing taking into account the physical and social environment of the animals, well organised colony management and correctly followed animal care regulations are indispensable prerequisites for animal experiments of high quality and reproducibility.

The husbandry shall provide a standardised macro- and micro-environment as basis of reliable and reproducible research results but should also take into account the welfare of the animals. Several recommendations have been published which serve as guidelines for proper (e.g. GV-SOLAS, 1988; ILAR, 1996; Jennings et al., 1998; van Zutphen et al., 2001), but not in any case comfortable (Sherwin, 2002) housing of mice. These guidelines refer to requirements on ventilation, temperature, humidity, lighting, noise levels, health status (see Chapter 27 on Health Monitoring; Nicklas et al., 2002), feeding, water supply, animal enclosures, handling and experimentation including

Appropriate conditions for breeding, maintenance and experimentation will be described with respect to the different hygienic levels. In animal experimentation the established (national) guidelines for the care and use of laboratory animals in line with the local animal welfare regulations have to be followed. For details, see the specific textbooks (e.g. ILAR, 1996; van Zutphen et al., 2001), but also Chapters 25 and 29 in this handbook. The LaboratoryMouse Copyright 2004 Elsevier ISBN 0-1233-6425-6

All rights of production in any form reserved

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anaesthesia, analgesia and euthanasia (see Chapter 34). The environmental requirements of mice are summarised in Table 24.1, the space requirements according to ILAR (1996) and the European standards (ETS 123, Appendix A presently under revision) are listed in Table 24.2. In general, the personnel in charge of handling the animals should be educated and well trained in the care of laboratory animals (e.g. according to FELASA (1995) guidelines). The standard procedures have to be strictly defined such as (i) hygienic procedures for personnel, (ii) daily monitoring of animals for adequate environmental conditions and general health, (iii) food (standard diet) and water control, (iv) regular changes of adequate cages and bedding, (v) cleaning and sterilisation of cages, water bottles, racks and other equipment, and (vi) sanitation programmes.

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aEuropean Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes, ETSNo.: 123,Appendix A (Draft, 2003), in the current version a minimum floor area of 200 cm2 is required (http'J/conventions.coe.int/t reaty/en/Treaties/Html/123.htm#APPENDIX-A).

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dMice are typically bred in male to female ratios of 1 : 1 or 1 : 2. If bred permanently monogamous (1 : 1),the male,female and any litter of pups may be kept together until the pups are weaned. In case of a trio (1 male and 2 females) kept in a micro-isolator cage, it is recommended that one of the two females be removed to a separate cage once observed to be pregnant.This facilitates compliance with housing standards and furthermore permits unobscured pedigree documentation. It is, however, permissible to keep the trio and the pups of one or two litters together providing that when the eldest litter turns 14 days of age there are no more than 12 pups in total in the cage (for reference see Reeb-Whitaker et al., 2001). Higher female ratios (1:3,1:4) are not recommended unless pregnant females are removed once observed to be pregnant.Weaning of pups is recommended by lIAR at 18-21 days of age especially when intensive mating is done where the male is kept with the female(s) permitting breeding on the postpartum oestrus. Weaning of mouse pups latest should be done by 28 days of age or if a new litter is borne by the still nursing female.

specified and for which the colony is regularly monitored (Nicklas etaL, 2002). Suggestions for the exclusion list as well as methods of monitoring can be found in Chapter 25 on Gnotobiology and Breeding Techniques and in Chapter 27 on Health Monitoring in this handbook. Quarantine originally used_to cover the potential latency period of infections by opportunistic or pathogenic agents, describes in this context a status in which, in particular, newly introduced strains from other instiTreating immunocompromised breeders with immunotutions have to be imported, maintained and put competent cells (from syngeneic donors such as an through repeated and thorough hygienic examination immunocompetent genetic background or F1 hybrids (screening for viral, bacterial and endo- ectoparasitic of the strain to be reconstituted and the immunocominfections) before the animals may be transferred into petent strain) can assist in the propagation of highly the respective animal quarters or the new strain immunocompromised strains (Wang et al., 1997; undergoes a rederivation process. Kawachi et al., 2000). For the reconstitution of nude Infectious denominates the status of animals either being mice, Foxnl"*, thymus homogenates can be injected infected naturally or within experimental research. intraperitoneally to reconstitute their T-cell defect. As a They are a risk for the other animals in the facility preventive measure for homozygous Prkdc saa, Ragl "1, and/or for humans. Animals with undefined microRag2~1, etc. mice an injection of syngeneic or F1 biological status are usually treated like infectious spleen cells i.p. (1-2 • 107), or bone marrow cells animals. (2-5 • 106) i.v. into juvenile mice has been shown to be very efficient (Mossmann, unpublished). This treatment improves the constitution of the individual mice and thus predestines those (however only) as breeders.

Preventive care for immunocompromised breeders

Animal facilities

Housing conditions Earlier descriptions of housing systems for small rodents have not lost their principal validity (see e.g. ILAR Committee, 1976; Spiegel, 1976; Otis and Foster, 1983; Heine, 1998) although many refinements have been introduced meanwhile. According to the animal house premises, the scientific demands, international standards and the risk of contamination within the building, different hygienic areas should be established. In principle, these could be:

Germfree or axenic, designating a status in which no microorganisms are present except those integrated in the genome. Gnotobiotic or gnotox~ic where the animals are colonised with a fully defined flora which may induce some resistance to ubiquitous microorganisms. This could be on the basis of a pre-stimulation of the innate immune response and/or on an interference between bacterial strains including yeast in the resident flora of the intestinal tract. Specified pathogen free (SPF) describes by definition the respective animals as being free from the pathogens

In the last years, individually ventilated cage (IVC) systems came into increased use. This new set up not only affects facility construction, but also animal care management and health monitoring. While about 20 m 2 proved to be optimal for rooms with the conventional cage system in scientific institutions 50-70 m 2 per room are recommended for an effective use of IVCs providing a maximum rate of cages respectively mouse holding capacity per square metre of floor space. In IVCs the ventilation of the single cage is severalfold higher in comparison to the conventional open caging, allowing some extension of the cage-changing interval, which may at least in part compensate for the higher labour intensity. If correctly designed, the use of IVCs considerably decreases the allergen and odour level in the animal rooms.

SPF-units By definition, animals are. free of specified pathogens. However, no declaration on residual microorganisms is given, implying the probability of extensive differences from one SPF-unit to another (Heine, 1980; O'Rourke

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etal., 1988; Boot etal., 1996). Therefore, it must be con-

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sidered that when transferring animals from one SPFunit into another, additional microorganisms may be introduced which can have an effect on the microbiological equilibrium. This is particularly to be expected if immunocompromised animals are being maintained (Ohsugi etal., 1996). SPF-units are either used as breeding stations, with restricted access for researchers, or for long-term experiments. These are protected by strict hygienic barrier systems with sterile supply of air, food, cages, bedding and any other material, while all personnel including researchers have to pass a water or air shower as entry lock (Otis and Foster, 1983; ILAR Committee, 1989; Heine, 1998). A conventional open caging system or IVC systems may be used within the SPF-unit. After microbial decontamination with e.g. formaldehyde or hydrogen peroxide (Krause et al., 2001), gnotobiotic animals can be introduced via a chemical entry lock. Ifstandardised diets are introduced by steam-sterilisation the diet has to be enriched ('fortified') with heat sensitive vitamins to ensure that sufficient amounts remain after the heat treatment. As an alternative, gamma-irradiated standardised diet (25 kGy) packed in waterproof evacuated

bags can be transferred in into the SPF-unit after sufficient disinfection of external surfaces. The drinking water should be sterilised either by heat, ultrafiltration, and/or fluorescent (UV) light and then acidified (using hydrochloric acid or acidic acid) to a pH of 3.0-2.5, or chlorinated at a pH of 5 using stabilized hydrochloride to 6--8 ppm free chlorine (Leblanc, pers. comm.) in order to minimise the microbial growth. Bedding material should be dust-free (<1% dust) and must be steam-sterilised prefilled in cages or separately in bags (Table 24.3). The personnel entering the barrier bring about the highest risk in terms of microbial contamination. Therefore, only well-trained and highly motivated staff (FELASA, 1995), having had no contact to external rodents for several days and being free of infections should be allowed to enter the SPF-unit. The microbiologicalstatus of the SPF-unit should be monitored on a regular basis (see Chapter 27 on Health Monitoring). All sick animals have to be removed from the unit (unless the disease status is part of the animal's genetic constitution or the experiment) and submitted to health monitoring (microbiological examination, necropsy). In addition sentinel (germfree or gnotobiotic)

FABLE 24.3: Suggestions for a u t o c l a v i n g t e m p e r a t u r e s and times

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animals should be checked at fixed intervals. Regular disinfection of floors, walls and racks should be a routine operation procedure in order to minimize the possibility of infections. If properly managed such units may be maintained free of unwanted microorganisms for many years. In case of an infection within the unit it is unlikely that this can be restricted to a few cages, if conventional caging is used. This may be prevented only by the use of IVCs when handled properly.

IVC systems The individual ventilation of the cages with HEPAfiltered air allows long-term bio-containment on the cage level when the handling of the cage's interior follows aseptic rules. Individually ventilated cage systems are used to breed and maintain animals in order to reduce the risk of miscellaneous microbial contamination and to improve environmental conditions, which may be of special interest when maintaining immunocompromised animals. Individually ventilated cage systems are particularly useful when a barrier-sustained (SPF) unit is not available and in experimental units, where easy access to the animals by the scientists is indispensable. In addition, IVCs with positive pressure are also ideal for a preliminary or timed containment of animals derived from different sources to maintain their respective hygienic status, if the area is barrier protected. For quarantine purposes without an additional barrier system and for infectious experiments these racks should be used with negative pressure. A problem of IVCs is the health monitoring requiring sensu str/ctu testing of each individual cage. Ventilation of the cages can be performed by blowers in the animal room. In this case the supply air is drawn from the room's air; the exhaust air duct should be loosely interconnected to the room's exhaust. For facilities to be newly constructed, separate channels for air supply/ exhaust for the room and the IVCs, respectively, might be installed. This avoids blower units within the room and allows decreasing room ventilation and bio-filter capacity and thus may reduce running costs. In addition, the stocking rate can be increased because it is no more limited to the ventilation capacity of the room. For most IVC-rack types the intra-cage pressure can be adjusted to be either positive or negative. This makes the units versatile in their use under different hygienic conditions. Ventilation with positive pressure in the cages counteracts the leakiness of the system. Negative cage-pressure may prevent the escape of microorganisms from the IVCs

and further reduces allergen escape. The different aspects of ventilated cage systems are described in an overview by Lipman (1999) and special topics are presented by Baumans eta/. (2002), Chaguri eta/. (2001), Clough eta/. (1994), Gordon et al. (2001), Hasegawa et a/. (1997), H6glund and Renstr6m (2001), Perkins and Lipman (1996), Reeb-Whitaker eta/. (2001), RenstriSm eta/. (2001), Tu etal. (1997) and Novak and Sharpless (2003). Different products are commercially available, in which the air is delivered either direcdy into the cage or distributed by a wide filter mesh in the hood to reduce the intracage air velocity. In many systems the exhaust air passes a filter in the cover to retain dust from the exhaust pipes. A critical event in IVCs is the failure of air supply either by an dectrical defect, blower break down or disconnection of tubes. It is therefore recommended to install an air flow controller in the supply air duct (positive pressure) or exhaust duct (negative pressure) respectivdy, which is connected to the alarm system of the animal house (Huerkamp eta/., 2003).

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The procedure of sterile handling of IVCs is labour intensive. This can be compensated - at least in part- by extending the cage change interval. Due to the higher intra-cage ventilation the ammonia concentration will be lower and the bedding stays drier for a longer period. In addition, increasing the change interval will reduce the stress for the animals (Duke et al., 2001) and may increase breeding efficacy (Reeb-Whitaker et al., 2001). The proper management of IVCs is critical and quite often underestimated. There are three different hygienic levels that have to be considered: (i) The sterility level of the autoclaved material: cage with bedding, lid and hood (sterilised as a not tighdy closed unit, or separately in a container or foil), diet (autoclaved or gamma-irradiated at 25 kGy) and water bottles, transferred sterile into the laminar flow changing station (e.g. within a filter hood covered cage); (ii) the hygienic level of the animal room (i.e. the direct environment of the animal enclosure termed 'room-clean'); and (iii) the hygienic status of the animals. In a correctly manipulated setting, these three levels have to be strictly discriminated. To maintain the health status of the animals within the individual cages it is advisable to run the regular caretaking routines by two animal technicians (a 'clean' and a 'room-clean' person; Table 24.4). In certain IVC systems with water bottles outside the hood, there is a high risk of contaminating the mice when bottlechange is not performed in connection with regular cage changing in the laminar flow cage changing station.

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1. Laminar flow cage changing station or laminar flow hood is running (30 min in advance). 2. A fast acting sterilisation compound (recommended:Clidox) is freshly diluted for gloves, bench worktop, (in separate beakers) for the forceps to handle the mice (alternately used) and a pincers to handle the lid. 3. The animal technicians handling the cages and animals should at least wear gloves; however it is recommended to also wear a surgical gown,cap and mask. Room-cleon person: The cage to be changed is placed into the changing station, the cage tag is removed and hood

and lid partially displaced backward. An autoclaved cage with bedding, lid and hood is placed into the bench and opened partially without contactingthe inner side of the cage. Clean person:Sterilised diet is transferred into the clean cage from the outside sterilised, irradiated food-bag. Sterile

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Static micro-isolators 'Static micro-isolators' are non-ventilated, partially perforated boxes with a tight filter medium, covering the perforation and a cover, which has to be sealed (Kraft, 1958). They provide a microenvironment, which is protected from adventitious contamination from outside. Micro-isolators are still in use (e.g. Han-Gnotocage) for the transport of germfree, SPF-founder and quarantine (Otto and Tolwani, 2002). They can also be used for short-term housing of germfree or gnotobiotic foster mothers (in a laminar flow cabinet). The disadvantage of these micro-isolators, however, is their impeded intracage ventilation which results in an increase in humidity, carbon dioxide and ammonia concentrations. With increasing animal density static micro-isolators become intolerable. However, static filter top cages prevent the release of allergens and are therefore well suited for transportation of mice within an institution.

Isolators (positive pressure) They consist of a closed construction with a germ-tight air inlet filter, a liquid air outlet trap or a germ-tight

outlet filter, long-arm gloves and a chemically sterilisable lock for the supply via an autoclave cylinder (see e.g. Trexler, 1983; Heine, 1998). Freshly mixed buffered peracetic acid (Kesla Pharma Wolfen, D-06803 Greppin, Germany), diluted to 2.0% is commonly used to sterilise the interior of the isolator and the entry lock. For details on how to generally operate isolators the reader is referred to Chapter 25 on Gnotobiology and Breeding Techniques, or Heine (1998). As a consequence of sterilisation for 30min at 134~ the diet may become less palatable (Porter and Festing, 1970) and masticable than a surrogate gamma-irradiated diet (50 kGy) supplied in evacuated bags.

Isolators (negative pressure) The equipment of most commercially available isolators allows the alternative use with negative pressure. In this version isolators protect the environment (other mouse colonies or man) from infections that are present in the isolator by a HEPA-filter in the exhaust valve. Their use is obligatory for experiments with high-risk pathogens. It is furthermore recommended for new strains from other sources being infected with agents imposing a high infectious risk to the existing colonies. All materials of isolators or quarantine have to be autoclaved when leaving the infectious area. In this

context, it should be mentioned that repeated autoclaving of cages with soiled bedding will destroy polycarbonate (macrolon), while polysulfone is much more resistant.

Quarantine

rooms, cages, lids, bottles etc. For drug dosages see Hawk and Leary (1995). It should be mentioned, however, that any treatment might interfere with the experimental outcome or might be associated with toxic effects (Scopets et al., 1996, Toth et al., 2000), as e.g. some knockout mice (targeted disruption of the multi-drug resistance gene) and CD-1 mice have been shown to be very sensitive to ivermecfin, with resulting mortality. Permanent antibiotic treatment of infections may induce bacterial resistance to antibiotics especially when used on a large scale. This will not only affect the animal colony (Hansen, 1995) by a shift in the gut flora (Morris, 1995) and other derangements in physiological functions (el Ayadi and Errami, 1999), it may lead to a contamination of the environment with multi-resistant bacteria that could be a hazard to humans. All bedding material of treated animals should therefore be sufficiently steam-sterilised prior to disposal.

As a consequence of the genetic manipulation, the exchange of breeding stocks between institutions has rapidly increased. Because of the presumably different hygienic constitutions, the single stocks should be preserved in their own microbiological status until rederivation can be performed. This can be achieved by the use of IVCs in positive pressure within a separate barrier-unit in negative pressure. Quarantine precautions should also be established when cellular material with unknown microbiological status has to be inoculated into experimental animals. If infected this biological material could be of risk for the animal facility (Yoshimura et al., 1997) contaminating the whole unit with a viral infection, such as ectromdia (Lipman et al., 2000), or mouse minute virus (Tietjen, 1992). Animals that have been gamma-irradiated should also be submitted to a quarantine-type area and not returned to their previous quarters (due to the same reasons). However, specific containers (sealed to avoid contamination of animals, with sufficient air volume to warrant The possibility of genetically engineering mice has oxygen supply for a certain period of time) have been brought about an exponential increase of new developed for irradition which may allow reintroduc- strains/stocks specifically constructed for the different tion of these mice after whole-body irradiation into a aspects of research. In general, donor mice ofoocytes or barrier unit. early embryonic stages are bought from commercial breeders. If these mice are maintained separately and manipulation of the oocytes/embryos (laminar flow, medium, washing, handling) is designed to interrupt a potential transmission of microorganisms, the risk of transmission of an infection should be low. Decisive for the hygienic status of the genetically manipulated progeny is, by far, the status of the foster mothers and The administration of therapeutics can influence the their handling being of special importance when creating outcome of animal experiments in different ways and immunodeflcient strains. should not become a routine procedure, nor is it a means to substitute for improved hygienic standards. Antibiotic treatment guided by microbial resistance testing may be a necessity in certain natural and induced mutants (e.g. Ncfl, due to their defective granulocyte bactericidal activity; Jackson et al., 1995) unless maintained under germfree or strict gnotobiotic conditions. The treatment of parasitic invasions is in particular dependent on the accompanying hygienic procedures e.g. use of gloves, Regulations contained in the ILAR (1996) GuideJbr the chemical and/or physical disinfection of the animal Care and Use of Laboratory Animals stress the importance

Presumptions for genetic manipulation

Therapeutic treatment

Identification systems

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of proper animal identification in sound research and humane animal care. The identification of single mice has become an indispensable tool even more so as a consequence of genetic manipulation, where genotyping results have to comply with the respective individual. Normally, correct labelling of the cages gives the first hint to strain, sex, age and individual numbering. The ILAR (1996) Guide as well as a number of other sources, list many acceptable identification methods for most common laboratory animal species. Several methods are being used: uJ U Z < z Z m

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Metal ear tags are an easy method of permanently marking laboratory animals. The procedure is simple using an ear punch and a specific applicator and does not require anaesthesia. The tags are available with a three digit numbering system up to 999. This implies a consecutive numbering of all mice, irrespective of the strain or a numbering of up to 999 within a strain. Ear tags with different colours per strain within a unit may be of help. Unfortunately, mice sometimes lose their tags, requiring remarking. Tattooing of footpads, tail etc. is an acceptable alternative, which may be especially helpful in marking new borne mice. Ear punching: Exterior markings of the pinna can be done on mice after two weeks of age without anaesthesia. Ear punch identification may be obliterated after several weeks because of wound healing or by fighting between individuals. A system of holes and notches permits a numbering from 1 to 9 respectively 10 to 90 on both ears (see Figure 24.1), allowing for an individual numbering system of up to 100 individual mice.

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Toe-clipping: One method of identification that has been used for rodents is toe-clipping. This method involves removal of the first phalanges of one toe of up to all four limbs, corresponding to a predetermined numbering code (ALAT Manual, 1998). The different digits removed code the identifier. Because toe-clipping is a potentially painful procedure that can also alter the gait or weight-bearing ability of a rodent's rear limbs, the ILAR (1996) Guide ~ r the Care and Use of Laboratory Animals limits its use only to justified instances (toe-clipping protocol must be discussed by the IACUC, North America), and requires anaesthesia for animals older than 1 week. In other countries this method is not permitted at all (e.g. German Animal Welfare Act w Transponder: The subcutaneous implantation of a transponder is without doubt the most reliable, but most expensive method for identification. This system is practically not limited in the number of individuals that can be differentiated and is rather feasible, when animals have to be identified very often, e.g. if monitoring of body weight or other parameters like body temperature have to be recorded together with the identification number (Kort et al., 1998).

Refinement Animal welfare regulations (see also Chapter 29 on Legal Regulations for the Protection of Animals Used for Scientific Experiments) require that the environment of the animals maintained should meet their physiological and behavioural needs and that the housing conditions must facilitate the performance of natural behavioural patterns and allow for adequate social contacts (Jennings et al., 1998). Current debate on enrichment of the animals' environment and space requirements has not come to a general conclusion. There is no doubt that cages with pregnant females should be equipped with nesting material like nestlets, paper towels etc. The published opinion is rather contradictory with respect to the effects imposed on research data obtained under 'standard' (sometimes called 'impoverished') vs. structurally and/or spatially enriched housing systems. While several groups report on distinct effects on behaviour, such as increased locomotion, exploration, learning ability, reduced anxiety etc. (Widman and Rosellini, 1989; Prior and Sachser, 1995; Boehm et al., 1996; Sherwin and Nicol, 1997; Powell et al., 1999; Sachser, 2001), other studies indicate

that an enrichment of the housing cage leads to an increase in aggressive behaviour (McGregor and Ayling, 1990; Haemisch and G~irtner, 1994; Haemisch et al., 1994) respectively to an increase in the coefficient of variation of several parameters depending on the strains analysed (Mering et al., 2001; Tsai et al., 2002, 2003). The latter result may suggest that due to a higher variation of different parameters from one experiment to another and between different environments the estimations of the appropriate number of animals to be used have to be increased. Marashi et al. (2003) report on an increase of aggressive behaviour in male mice and elevated levels of stress hormones in mice housed in structurally (plastic inset and a wooden scaffolding) and a structurally as well as spatially enriched environment (richly structured by a complete, passable enriched cage, extra plains, plastic stairs, wooden footpaths, hemp ropes, and a climbing tree). Interestingly, it is concluded that stress is a most important prerequisite to achieve good welfare. This controversy with a thus far agreed notion that animals should be housed with the goal of maximising speciesspecific behaviour and minimising stress-induced behaviours needs further analysis. For a detailed bibli-

ography on mouse enrichment and refinement see the UCCAA database providing access to regularly updated information that can be addressed to improve or refine research with mice (http://www.vetmed. ucdavis.edu/Animal_Alternatives/miceR3.htm).

Genetic monitoring Mutations as well as differential fixation of alleles at early generations of inbreeding may alter the genetic constitution and thus the phenotype of an inbred strain. Many of the phenotypic differences detected between substrains have been shown to be due to these factors. Inadvertent outcrossing will alter a strain seriously, questioning its further use for research, since results are no longer comparable and repeatable. It is thus of utmost importance to separate strains that are not immediately distinguishable by their phenotypic appearalace. In most facilities due to shortage in shelf space and separate animal rooms, many strains have to

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outside the cage should be isolated, if identification is possible, otherwise killed. 3. Cages and hoods should be in sufficient condition that no animal can escape or enter another cage, a problem more often encountered in mouse than in rat breeding units. 4. For ease of identification and in order to prevent an inadvertent mix-up, cage tags should have a strain-specific colour code and a strain-specific number (code). 5. Cagetags should always be filled out properly, including the strain name, strain number, identification numbering of the animals in the cage, parentage, date of birth, generation, and in case of experimental use the name and licence number of the scientist.

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6. Ifa cage tag is lost, one should not redefine the cage except in the case of definite proof of identity through marked animals within the cage. 7. If at weaning the number of animals is larger than that recorded at birth the whole litter should be discarded or submitted to genetic monitoring. 8. Any change in phenotype and/or increase in productivity should immediately be reported to the colony supervisor. The latter change should always be considered suspect for a possible genetic contamination. 9. Regular training programmes on basic Mendelian genetics, systems of mating and the reproductive physiology of the animals maintained should make animal technicians and caretakers conscious of the consequences any mistake will impose on the colonies. Further training should stress the importance of a search for deviants as potentially new models for biomedical research.

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be housed in one room, making regular screenings for housing unit. There are numerous publications and strain discriminating markers as well as the differentiat- textbooks with protocols for polymerase chain reaction ing locus (in case ofcongenic strains) indispensable. (PCR) amplification and electrophoretic separation of As repeated handling of animals during regular care- the amplicons. Moreover, commercial suppliers of taking cannot be avoided, there is always the risk of mis- primers (e.g. Research Genetics) and of genetically takes. Proper colony management is the first step modified animals (e.g. The Jackson Laboratory's towards the provision of authentic laboratory animals. Induced Mutant Resource genotyping protocols, Either inadvertently an animal might be placed into a http://www.jax.org/imr/geno_index.html) do provide wrong cage, or a false entry is put on the label. Assigning PCR protocols. In any laboratory setting, it might be this type of work to well trained and highly motivated necessary to adjust temperature conditions as well as animal technicians should be a matter of course. This Mg ++ concentrations for each microsatellite marker. can be summarised in nine principles of a proper colony For routine screening separation in agarose gel and management (see Table 24.5). The colony set-up and visualisation by ethidium bromide will suffice. If sepastructuring with nucleus colonies in a single (Festing, ration of the amplicons is insufficient one should run a 1979) or a (modified) parallel line system (Hedrich, polyacrylamide gel electrophoresis. Instead of radioac1990), pedigreed expansion colonies and multiplication tive labelling with 32p using a kinase reaction and since colonies should be self evident, but strictly monitored. the half-life of the isotope is relatively short, a silver There are several publications dealing with the set-up staining procedure is recommended. Information on of colonies for maintenance and large-scale produc- restriction fragment length polymorphisms (RFLPs) as tion (Green, 1966; Lane-Petter and Pearson, 1971; determined by a Southern blot (Sambrook eta/., 1989) Hansen etal., 1973; Festing, 1979). In general, perma- using a specific probe is partly provided through mouse nent monogamous mating is to be given preference, as genome informatics (MGI) maintained by The Jackson this provides a constant colony output by minimal dis- Laborat6ry. turbance of the litters during the early post natal period Despite the ease of analysis and the abundant and by utilising the chance that females are fertilised at number of markers, classical methods are still relevant the post-partum oestrus. and may need to be verified, and sometimes may even The measures required for genotyping a strain have allow for a faster and less expensive phenotyping. In this to be adjusted to the specific needs and may depend on context immunological markers are of prime importthe scientific purpose, the physical maintenance con- ance. This group is composed of cell surface markers, ditions and the laboratory equipment. Nevertheless, such as the major histocompatibility antigens (H2), there are specific demands (although unfortunately not lymphocyte differentiation antigens, red blood cell stringent rules) on how to authenticate a strain or to antigens, minor histocompatibility antigens, allotypes verify its integrity. (immunoglobulin heavy chain variants) which can be For any authentication it is necessary to determine determined using specific antibodies by Trypan blue dye a genetic profile that is to be compared with published exclusion test, flow cytometry, immunodiffusion and data (as far as available), and which allows to distin- enzyme-linked immunosorbent assay (ELISA). Further guish between (all) strains/stocks maintained in one methods that can be applied easily and determine a speunit. In general this profile is composed of mono- cific phenotype may also be used, as e.g. the lysosomal genetic polymorphic markers, which may be further trafficking regulator (Lyst~, beige, expressing a pigmendifferentiated by the method of detection into tation and platelet storage pool defect). The phenotype immunological, biochemical, cytogenetical, morpho- of homozygous beige mice can be demonstrated by logical and DNA markers. Microsatellite markers a prolonged bleeding time (20min in homozygous (Simple Tandem Repeats, STRs) have almost fully Lyst bgvs. 6 min in unaffected wild-type or heterozygous replaced the dassical genetic markers in routine appli- controls), or a histochemical staining (checking for cations. A large number of primer pairs is available abnormal giant lysosomal granules detectable in all tison the world wide web (e.g. through Research Genetics sues with granule-containing cells; see Novak et aL, Inc., Huntsville, AL, USA, http://www.resgen.com). 1985). However, as with the classical markers it is indispThe set-up of a profile is time consuming and ensable to set up a genetic profile representing a ran- expensive, but strongly recommended as an initial check. dom sample of the genome, which should be evenly In case of a segregating background genetic profiling is spaced on the chromosomes, and which will allow to pointless, as the typing results will only assist in deterdiscriminate between all strains maintained per separate mining the degree of heterogeneity. However, these

results may provide hints on modifying genes, if the stock is incipient inbred and nearly homozygous. In case several strains with an identical coat colour phenotype, but different genetic modifications, are maintained in one room, all (induced) genetic modifications need to be tested for by PCR or other appropriate methods. Still one needs easy measures to distinguish between strains that are co-maintained and for which it is important to dearly identify an outcrossing event. A critical subset of the markers, at least the differentiating (genetically modified) markers for a given strain panel will provide reasonable information on the genetic quality of a strain and may partially serve to authenticate each strain within the unit. Unfortunatdy, with each strain added to such a unit the number of markers in the critical subset increases. These critical subsets need to be verifed at regular intervals (every 3-6 months). The intervals and the number of animals to be tested is increment to the number of strains co-maintained and to the size of each colony. Irrespective of these methods one of the most powerful information on an inbred strain is the demonstration of its isohistogeneity, only demonstrated by skin grafting. The technique is easy to perform, but time consuming due to an observation period of 100 days (for a description of the techniques see Hedrich, 1990). In certain immunodeficient mutants (e.g. Foxnl", Prkdc *aa, Raglt% Rag2TM) a direct demonstration of isohistogeneity is impossible, as these animals are incapable of mounting an allorecognition response. Transferring grafts from these immunodeficient animals to their syngeneic background strains will circumvent this.

Genetic problems in engineered strains Due to the enormous number of genetically modified strains their phenotypic discrimination is fairly impossible. In addition to the risk of mixing identities in the course of cage changing, false genotyping or false identification of the animals may be a source of mistakes. Therefore, regular screening ofhomozygous or still segregating strains/stocks is indispensable. In the process ofbackcrossing a transgene or targeted mutation onto a defined background it is advisable to select and analyse markers sufficiently differentiating between the background strain and the donor stock of the genetic modification. A genetic monitoring using markers similar to

a marker-assisted selection protocol (speed congenics production; see: Markel et al., 1997, Wakeland et al., 1997, Visscher, 1999) is recommended.

Computer assisted management of animal facilities The production and breeding of gene-manipulated mouse strains in most institutions has exponentially increased in recent years. In addition, the need to document animal breeding and experimentation for governmental and scientific purposes is also increasing. A computer database can be very helpful to facilitate the efficient management of this information. With such a tool, it is possible to maintain an overview of many aspects of breeding management such as: mouse strains, breeding status and availability, services provided, registered users and ethically reviewed projects (e.g. by the Institutional Animal Care and Use Committee, IACUC). The database should be multi-user compatible, and platform-independent within the computer network of the institution and should be designed in such a manner, that it helps the facility administrator, researchers, animal technicians and the animal welfare officer to maintain an overview of the work, as well as to support communication with each other and to reduce the workload. Typical functions of such a database could include: Mouse strains:. Data of all (sub)strains and sublines maintained using proper nomenclature as well as internal laboratory denominations and a coding system, strain owner, responsible investigator, genotype, phenotype, breeding information, breeding/holding unit etc. can be stored. An ordering system would allow users to submit jobs to the breeding facilities, such as ordering of animals for experimentation or shipment, ordering of tissues, cryopreservation, immunisation, embryo transfer, isolator breeding etc. The animal technicians in the breeding facilities could process these jobs and the status of each job should be viewable at any time. Animal welfare Projects approved by governmental authorities for work with experimental animals could be stored, as well as the type of experimental procedures permitted, the number of animals allowed, registered IAuthor of this paragraph: Peter Nielsen. Max-Planck-Institut ftir Immunbiologie,Freiburgi.Br., Germany.

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persons etc. User list: All users working with animals could be stored, along with strain access information. Breeding analysis: It should be possible to register the litters and offspring for all breeding for each strain. This information permits a clear overview on all breeding including animal and cage inventory, sex, phenotype, genotype, genealogy etc. For the purpose of archiving or further analysis, the data should be exportable to other programmes like MS Word | or MS Excel | Inventor~ It could be useful to know, at any time, the number of animals or cages present in the breeding facility. This can be useful for allocating breeding and holding space, as well as for cost calculations. Invoices:. If desired, it should be possible to create invoices for animals, cage or special animal care costs, for jobs (specific diagnostics, sample collection etc.), as well as for other services rendered. Online information: Data sheets or comments could also be integrated, which describe the use of the database and aspects of animal work, some of which may be specific for the institute. Language preference. The employees in many research institutes may not have a common mother language. For this reason, it could be useful if the database can accommodate a preferred language for each user. Such a database should help to maintain an overview of experimentation with animals. It should stimulate an efficient communication between the different usergroups and save world Researchersshould be able to see which strains and ethically reviewed projects they are responsible for, including the current number of animals used and the status of all jobs they have submitted. They may also be allowed to run a detailed analysis ofgenotype frequencies, pedigrees, reproductive data etc. Animal

technicians could check which animals have been ordered for a specific date and where (holding unit/laboratoty) these should be delivered/shipped. Animal welfare officers should be able to verify how many animals have been ordered for which purpose, the duration of the treatment etc. They could use this information to easily create a report providing a summary of the animals used in any experiment. This report may be submitted directly to the government authorities, if required. Breeding administrators can identify which animals belong to whom and how many mice have been used. They can thus insure that sufficient numbers per sex and per strain will be available within the institution. If desired, invoices for the services provided could also be created. For additional information, several databases, which have been developed for colony management, animal breeding and experimentation are listed above.

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Renstr~m, A., BjOring, G. and HOglund, A. U. (2001). Lab. Anita. 35, 42-50. Sachser, N. (2001). In Copingwith Challenge:Welfarein Animals Including Humans (ed. D.M. Broom), pp. 31-48. Dahlem Workshop Report 87, Dahlem University Press, Berlin. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York. Scopets, B., Wilson, l~P., Grifflth, J.W. and Lang, C.M. (1996). Lab. Anita. Sc/. 46, 111-112. Sherwin C.M. (2002). In Comfortable Quarters fir Laboratory Animals. (ed. V. Reinhardt and A. Reinhard0 pp. 6-17. Animal Welfare Institute, Washington, DC. Sherwin, C.M. and Nicol, C.J. (1997). Anim. Behav. 53, 67-74. Spiegel, A. (1976). Versuchstiere: Eine Ein~hrung in die Grundlagen ihrer Zucht und Haltung. Gustav Fischer Verlag, Stuttgart. Tietjen, R.M. (1992). Lab. Anita. Sci. 42, 422. Toth, L.A., Oberbeck, C., Straign, C.M., Frazier, S. and Rehg, J.E. (2000). Contemp. Top. 39(2), 18-21.

Trexler, P.C. (1983). In The Mouse in Biomedical Research (eds H.L. Foster, J.D. Small and J.G. Fox), pp. 1-16. Academic Press, New York. Tsai, P.P., Pachowsky, U., Stelzer, H.D. and Hackbarth, H. (2002). Lab. Anita. 36, 411-419. Tsai, P.P., Oppermann, D., Stelzer, H.D., M~flller, M. and Hackbarth, H. (2003). Lab. Anita. 37, 44-53. Tu, H., Diberadinis, L.J. and Lipman, N.S. (1997). Contemp. Top. 36(1), 69-73. van Zutphen, L.EM., Baumans, V. and Beynen, A. (2001). l~nciples of LaboratoryAnimal Science. Elsevier,Amsterdam. Visscher, P.M. (1999). Genet. Res. 74, 81-85. Wakeland, E., Morel, L., Achey, K., Yui, M. and Longmate, J. (1997). ImmunoL Today 18, 472-477. Wang, B., Simpson, S.J., Hollander, G.A. and Terhorst, C. (1997). ImmunoL Rev. 157, 53-60. Widman, D.R. and Ra)sellini, R.A. (1989). PhysioL Behav. 47, 57-62. Yoshimura, M., Endo, S., Ishihara, K., Itoh, T., Takakura, A., Ueyama, Y. and Ohnishi, Y. (1997) Exp. Anita. 46, 161-164.