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MANAGEMENT PROCEDURES QUARANTINE AND CONDITIONING Following are guidelines for operating a quarantine program: Upon arrival at the facility, primates should be examined, preferably by a veterinarian with training and experience in laboratory-animal medicine. Newly arrived primates should be quarantined for 30 to 60 days before being added to the resident colony. The period of quarantine depends on the source, species, and condition of the animals on arrival. The quarantine period should be extended upon diagnosis of certain infectious diseases. Animals in quarantine may be housed individually or in pairs. Housing of large groups in gang cages during the quarantine period is not recommended. Daily observations should be made by a person trained in recognizing signs of disease. All abnormalities should be reported daily to the veteri- narian or individual responsible for animal health. Animals showing signs of illness should be removed from contact with healthy animals. Immediate steps should be taken to diagnose the illness, and necessary treatment instituted. The primate quarantine areas should be used to house primates only. Personnel working in quarantine areas should wear distinctively colored garments and should be restricted to these areas for the entire work period. If this is not possible, the conditioned animals should be cared for before the animals in quarantine. Personnel should change clothes before entering other areas of the colony. Personnel working in quarantine areas should be required to wear pro- tective garments, including cap, mask, gloves, and protective footwear, as directed, and to observe all other prescribed safety precautions. All handling equipment, such as nets, transfer boxes, and gloves, should not be removed from the quarantine area, and should be sanitized daily. 22

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23 Presence of certain infectious diseases may require daily sterilization of equipment. Personnel with recognizable respiratory or gastrointestinal ailments should have limited contact with primates. The number of animals received in one shipment should not overtax the staff. Several small shipments received over a period of time are more de- sirable than a single large shipment. After the primates have completed the quarantine period, it is desirable to add a conditioning period before using them in research. The purpose of the conditioning period is to further stabilize metabolism, nutrition, and health, and to improve the animals' suitability as biological models. The length of this period will depend on the research requirement for which an animal will be used. COLONY MAINTENANCE Colony-maintenance procedures have a purpose similar to that of facilities. They are intended to provide the animals with the care and comfort they cannot provide for themselves in a laboratory environment. Environmental Control VENTILATION Ventilation should be considered in relation to the density of the animal population, the species of animals housed, and the nature of the laboratory activity. Ten to fifteen changes of fresh, ade- quately diffused air should take place each hour. The effect of air movement on an animal's body-surface temperature must be considered in relation to cage design and location. Sudden changes in temperature, drafts, pockets of dead air, and sizable gradients of tempera- ture from floor to ceiling are not permissible. Recirculation of air is not recommended. A mechanical ventilating system is necessary in most indoor primate facilities. TEMPERATURE The temperature maintained in the animal rooms should be appropriate to the species. If animals are routinely main- tained outdoors, provision should be made for a heated, sheltered area to which the animals may have access when the temperature falls below com- fortable levels. High- and low-temperature alarm systems are valuable for the animals'

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24 protection, and graphic records are useful for determining system perfor- mance. A diurnal pattern of lighting with a minimum of 12 hours of light per day is recommended. Feeding Food should be available daily unless otherwise indicated for health or sci- entific reasons. It should be palatable and of sufficient quantity and nutri- tive value to meet the normal daily requirements (Nutrient Requirements of Laboratory Animals, NAS-NRC Pub. 990, National Academy of Sciences- National Research Council, Washington, D.C., 1962) for the condition, size, and species involved. Food receptacles should be accessible to all animals and located so as to minimize contamination by excreta. They should be cleaned and sanitized regularly to prevent the food from molding, deteriorating, or caking. Watering Potable water should be readily accessible at all times or offered at least twice daily, except where otherwise indicated for health or scientific rea- sons. Preferably, water receptacles should be of the closed type, such as water bottles with sipper tubes, or automatic watering devices. Water bottles should be sanitized at least weekly. If open receptacles are used, they should be sanitized daily. Sanitation DAILY CLEANING OF CAGES Excreta should be removed from cages at least once daily. When hosing or flushing methods are used for this purpose, precautions should be taken to keep the animals dry. Care should be taken to prevent splashing of material into adjacent cages. If drop pans or troughs are used, they should be washed daily. SANITATION OF CAGES Cages should be cleaned and sanitized before new animals are introduced, and often enough to prevent an accumulation of debris and excreta. (See Federal Register, Vol. 32, no. 37, Part II, p. 3280, United States Department of Agriculture, regulations for Public Law 89-544.)

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25 HOUSEKEEPING Premises (buildings and grounds) should be kept clean and in good repair in order to prevent injury to the animals and to facilitate prescribed husbandry practices. Premises should remain free of accumulations of trash. VERMIN CONTROL An effective program for the control of insects and avian and mammalian pests should be in operation. PERSONAL HYGIENE Facilities to maintain cleanliness among animal colony personnel should be provided. Clean clothing suitable for use in the animal facility should be worn. Type and Number of Employees A sufficient number of employees should be engaged to maintain the pre- scribed level of husbandry practice. Supervision should be provided by persons having a background in animal husbandry or care. This qualification may be satisfied by completion of an animal-care-technician course or by equivalent practical experience in animal care. ARTIFICIAL REARING This section applies to rearing Old World monkeys and baboons. Informa- tion on rearing chimpanzees can be obtained from the Delta Regional Pri- mate Research Center, Covington, Louisiana 70433; the 6571st Aeromedical Research Laboratory, Holloman Air Force Base, New Mexico 88330; and the Yerkes Regional Primate Research Center, Emory University, Atlanta, Georgia 30322. Infant Care The mother of a newborn primate usually eats the placenta and membranes and in the process cuts the cord and cleans the infant. If the infant is strong and vigorous, it is usually able to cling firmly immediately postpartum. Normal births usually take place at night. Infants derived by Caesarean section from anesthetized mothers are likely to be depressed. They should be resuscitated promptly. Removing the infant from the mother can result in injury to either or

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26 both. Care must be taken. With the chimpanzee and baboon it is usually necessary to sedate the mother. Phencyclidine hydrochloride* is satisfactory for this purpose. Upon removal from the mother, the infant is weighed and examined for defects, and the cord is wiped with antiseptic and tied. At this time the in- fant may be identified by a tattoo number. Newborn baboons weigh 500 to 1,000 g, and rhesus monkeys 375 to 550 g. Pig-tailed macaques, Japanese macaques, and Celebesian apes are in a weight range similar to that of rhesus monkeys, but they tend to be heavier. Crab-eating macaques and vervets weigh about 100-150 g less than rhesus monkeys. A weight loss of 10 percent usually occurs during the first 36 hours after birth. Normal, healthy infants usually regain their birth weight by 5 days of age. During the first 60 days, Old World primates attain a weight almost double their birth weight; baboons increase their weight by about 60 percent. The first 48 hours after birth are critical to the infant's survival. The in- fant's clinical condition is assessed most reliably by its weight and amount of fluid intake. The color of the mucous membranes and skin, character and rate of respiration, heart rate, and change in weight also can be used to de- termine the infant's condition. Food intake, character and frequency of stool, and behavioral observations also are important and should be recorded daily until the infant is weaned. After 30-60 days of age infants should be placed together in a play cage for at least 2 hours daily. The larger the group the better. Physical contact among infants is important in establishing normal social relationships. Housing For the first 15 days of life the infant is housed in a standard human incu- bator or a germ-free isolator. Temperature is maintained between 88 and 92°F and relative humidity at 45-50 percent. The primate infant has poor thermostability; even after it is 2 weeks old, its temperature will tend to approach ambient temperature. Sudden drops of more than 2 or 3°F should be avoided. The incubator can be divided into two compartments (Figure 5), each with a surrogate (a cloth pad to which the infant clings, as it would to its mother) and a bottleholder for ad libitum feeding. At about 14 days of age, strong, vigorous infants should be transferred *Sernylan (Paike-Davis).

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27 FIGURE 5 Converted human incubator. to cages about 18 in. wide, 18 in. long, and 24 in. deep (Figure 6). They should be caged singly to permit individual observation. Each cage should contain a surrogate and a bottleholder. Room temperature should be maintained at 85°F. Feeding and Weaning Infants are fed at first from 4-oz and later from 8-oz human-infant nursing bottles, which should have the standard rubber premature-infant or regular nipple. A doll bottle and nipple is often more effective for feeding infants of the smaller species for the first few weeks. Bottles and nipples should be sterilized with the same care that is exer- cised in sterilizing for human infants, and high standards of cleanliness should be maintained. Cleanliness minimizes the danger of diarrhea and pneumonia, which are the most commonly encountered infant infections. Bottle-feeding is accomplished in three stages, discussed below. The formulas should be prepared daily in bottles. Tap water should be used as the diluent. Bottles containing newly prepared formula should be sterilized. They should then be cooled and kept in a refrigerator until the formula is

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28 FIGURE 6 Infant cage fed. Usually the formula is warmed to about body temperature before it is fed. First Stage The infant should receive its first feeding 6 to 12 hours after birth and subsequent feedings at 2-hour intervals for the next 24 hours, ex- cept during the period from 10 p.m. to 6 a.m. In these first feedings the diet should consist of dextrose or glucose solu- tion (5 to 10 percent), and it should be fed ad libitum. For an Old World monkey, the amount of intake per feeding ranges from 10 to 15 cc; for a baboon, from 15 to 25 cc. Second Stage From day 2 the infant receives a commercial human-milk- substitute baby formula.* As before, the formula is fed every 2 hours except during the period from 10 p.m. to 6 a.m. For the first 3 days of this stage, the formula should be fed with a 10- percent dextrose solution in water at a ratio of 1:1. By day 4 the formula should be fed full strength to Old World monkeys; the 1:1 ratio should be continued for baboons until weaning. Between the second and the fifth day, a strong, vigorous infant can be started on a self-feeding, ad libitum feeding schedule. Every 2 hours (nine *SIMILAC (Ross Laboratories) or SMA-S26 (Wyeth Laboratories).

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29 times a day) the infant is given a bottle containing 15 to 35 cc of formula, the amount depending on the infant's size. As the bottle is placed in the holder (see the preceding discussion of housing), a gentle tapping noise is made to help the infant locate the bottle. Many nurseries continue hand-feeding after the first 3 to 5 days of life, feeling that burping is essential after each feeding to prevent vomiting. Burping the infant is accomplished by placing the infant in the erect posi- tion, gently tapping and massaging its back after it has consumed several cubic centimeters of formula. VARIATIONS IN FORMULAS The basic formulas are (1) dextrose or glucose solution and (2) commercial human-milk-substitute baby formula. These are referred to under "First Stage" and "Second Stage." Experienced laboratories have successfully reared Old World monkey in- fants on a formula in which the standard concentration of 20 calories per fluid ounce was followed. Unpublished data* suggest that a standard formula diluted 1:1 with tap water is preferable for baboons because it reduces a tendency toward electro- lyte imbalance that occurs in this species when the full-strength formula is fed. The calorie deficit is made up by adding glucose or dextrose or by in- creasing the total volume of formula to be consumed. In addition, from 8 to 12 weeks of age until weaning, a baby cereal (35-percent protein) is added to the formula. Third Stage After 5 days of feeding at 2-hour intervals (second stage), infants should be fed at 4-hour intervals, five bottles per day, until weaning. At each feeding, an Old World monkey receives 20 to 80 cc of the same for- mula that was fed in the second stage; a baboon, up to 120 cc. Throughout bottle feeding, adequacy of intake should be judged by daily rate of gain. Satisfactory rates of gain are different for different species. Figure 7 shows growth rate for the rhesus infant. In addition to formula, each infant should receive 1/4 cc of a standard pediatric multivitamin preparation with an iron additive (where this is not provided in the formula) every other day until it is weaned to adult food. *W. R. Voss, Director, Primate Colony, Department of Virology, Baylor University College of Medicine, Houston, Texas.

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30 . » If It 15.0 10.0 5.0 175 235 295 355 415 475 CONCEPTIONAL AGE (DAYS) 535 595 FIGURE 7 Growth rate (body weight) of infant rhesus macaque monkeys (Macaca mulatta) from birth to 595 days conceptional age (average of 10 animals). It is desirable to continue the vitamin supplementation until the infant is 6 months old. When they are about 30 days old, infants may be taught to accept milk from a pan; teaching must be gradual. Generally, the animals have their in- cisors at this age, and solid food can be introduced into the diet. When they are about 6 months old, they may be placed entirely on an adult feeding schedule. Basically, weaning consists in decreasing the milk intake and increasing the intake of solid food, such as commercial monkey diet and fruit, and maintaining adequate daily caloric intake while the change is in progress. The caloric intake of rhesus infants is given in Figure 8. If quantities are adjusted to correspond with differences in the weight of the animals, requirements stated in the figure can be applied to baboons. The commercial monkey diet is more acceptable if soaked in formula at first. DISEASE CONTROL General Frequent inspection, periodic examination, and sanitation are important factors in disease control. All animals should be observed daily for signs of illness. The procedures to be followed when such signs are noted are stated under "Quarantine," page 22.

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31 v 250 r SI 3 * 2 £ II *! 200 \so too \7S 235 295 355 4I5 CONCEPTIONAL AGE (DAYS) 475 FIGURE 8 Caloric intake of infant rhesus macaque monkeys (Macaca mulatta) from birth to 505 days conceptional age (average of 10 animals). Personnel should not be allowed in an area to which they have not been assigned without permission from a person responsible for animal health. A dead animal should be removed as soon as discovered, placed in an impervious container, refrigerated, and held for necropsy examination and ultimate incineration. Tuberculosis Tuberculosis is frequently encountered in colonies of Old World monkeys. New World monkeys are less susceptible but should be examined frequently. Newly arrived animals must be tested on arrival and at 2-week intervals until each animal in the group has passed 3 consecutive negative tests. The intradermal palpebral test is customarily used to detect tuberculosis. Usually 0.1 ml of Koch's Old Tuberculin (KOT) containing 15 mg of KOT or the com- parable purified protein derivative is injected. The injection site is examined at 24,48, and 72 hours. A positive reaction is indicated by redness and swell- ing at the injection site, frequently involving the entire eyelid and accom- panied by a profuse exudate. Routine radiographic examination of the chest of each animal on arrival is encouraged to detect advanced cases of tuberculosis which may be tuber- culin-negative. Immediate elimination of all positive reactors is recommended, followed

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32 by necropsy and histopathological diagnosis. Testing should be repeated at 1- to 3-month intervals after the initial quarantine period. See Appendix I, "Suggested Standard Procedures for Detection of Tuberculosis," p. 44. Pneumonia Pneumonia is commonly encountered in primate colonies. It is generally associated with improper environmental control and with the stress induced by capture and transportation. It is primarily a problem during the first month after arrival and is seldom a problem in well-conditioned animals that are properly housed. Pulmonary Acariasis (Lung Mites) Pulmonary acariasis is commonly encountered in wild-born Old World mon- keys, particularly macaques. The effect of the parasite on the health of the animal is considered to be of minor significance unless the respiratory sys- tem is heavily involved. Enteric Diseases Enteritis is probably the most common of the diseases affecting laboratory primates. Major causative organisms are Salmonella, Proteus, and Shigella. Other intestinal pathogens, including protozoa, helminths, and viruses, may be responsible for various intestinal disorders. Many enteric disorders are related to lowered resistance resulting from the stress of capture and transportation, to unfamiliar diets, and to unfa- miliar colony confinement. Good husbandry practices are essential to the prevention and control of enteric disorders regardless of their cause. SAFETY AND SECURITY Construction in all primate areas should be designed to prevent the escape of animals. A vestibule, equipped with self-closing doors at all access points, should

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33 intervene between cages and the outside doors of the buildings. The vesti- bule doors should have locks. Cages should be properly constructed (discussed under "Cages and Equip- ment," p. 11). The primate areas should be completely enclosed. Equipment and procedures must insure the safety of personnel, must be noninjurious to the animals, and must minimize the possibility of trans- mission of infection from animal to man, animal to animal, and man to animal. Restraint of animals should be both effective and gentle, with emphasis on protecting animals and handlers from injury. The canine teeth may be surgically removed from large primates that are aggressive and require frequent handling. Clipping off the ends of the canine teeth frequently results in the formation of abscesses and is not recommended. PERSONNEL MANAGEMENT The reliability of biomedical data obtained from laboratory primates depends largely upon sound preventive medicine practices, rigid environmental control, and the services of highly motivated and properly trained animal technicians. References and source materials for technician training are available from the Institute of Laboratory Animal Resources, National Research Council, 2101 Constitution Avenue, Washington, D.C. 20418, and from the American Association for Laboratory Animal Science, P.O. Box 10, Joliet, Illinois 60434. A correspondence course in basic animal care may be obtained at a nominal cost from the Ralston Purina Company, 835 South 8th Street, St. Louis, Missouri 63102. The laboratory veterinarian should assist tech- nicians in interpreting and applying the information in these materials and should provide supplementary training and guidance. Animal Handlers SUITABILITY AND HEALTH Animal handlers must be strong, agile, highly motivated, and emotionally fit to work with primates. They must have an understanding of the animals' needs and must feel concern for their humane and proper care. They should be in good health. New employees should have a complete pre-employment physical examination, which should include chest x-ray, tuberculin test, and stool culture.

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34 The fact that certain of the diseases occurring in primates are transmis- sible to humans makes it necessary to give continuing consideration to employees' health. Employees should not eat, drink, or smoke in the animal areas. Employees should have physical examinations annually and chest x-rays and tuberculin tests semiannually. An immunization program should be maintained for all employees. Employees should not work in animal areas when they are ill. All bites, scratches, or other injuries should receive prompt medical care and should be reported to the designated person. TRAINING New employees should receive training in primate handling and management. This training should be continued and should stress both animal and personal health considerations. EQUIPMENT Appropriate clothing should be provided for personnel working with primates. Freshly laundered clothing should be available daily. The following should be worn in animal areas: shoes or boots that can be washed and sanitized, disposable face masks, caps, and shields or other eye protection, as indicated. Protective gloves should be worn, preferably with attached sleeves to the shoulder for arm protection, for catching and restraining monkeys. PRECAUTIONS B virus infections are found mainly in the Macaca mulatta, but other Old World species have been implicated. When transmitted to man, B virus infec- tion usually produces a fatal encephalomyelitis. The danger to persons work- ing with M. mulatta or its tissues should be recognized. All primates, especially those newly received, should be viewed as possible vectors of a number of diseases transmissible to man, such as tuberculosis, the dysenteries (salmonellosis, shigellosis, amebiasis), B virus, yellow fever, and infectious hepatitis. A physician should be consulted for appropriate prophylactic guidance. NUTRITION The nutritional needs of many species of primates can be met with com- mercially available diets. Vitamin, mineral, and protein supplementation

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35 may be indicated for infants, breeding animals, nursing mothers, and ani- mals in an undernourished or otherwise poor condition. Some species re- quire constant supplementation. Fresh water should be available to the animals at all times. Tree Shrews Tree shrews, essentially omnivorous, have been successfully maintained in captivity on commercial monkey and dog diets and also on diets consisting of a wide variety of fruits, vegetables, insects, and meats. Maximum food consumption is stimulated when moderate portions of of fresh food are offered at regular intervals during the day. Tupaia infants have been successfully hand-reared from birth on the fol- lowing formula: 2/3 pint whole cow's milk, 1/3 pint evaporated milk, 1/2 raw egg yolk, and 1 teaspoon corn syrup. Feeding of 4 or 5 g of this formula every 8 hours through the first 3 weeks has been recommended. Semisolid foods have been introduced at 4 or 5 weeks of age. Lorisidae The Lorisidae are primarily carnivorous and insectivorous; they occasionally eat fruits and leaves. In captivity they can be maintained satisfactorily on commercial monkey diets. Transition to such diets should be made gradually. Smaller species may have difficulty in chewing hard biscuits; this can be overcome by soaking the biscuits in water. Since the Lorisidae are nocturnal, fresh food should be placed in the cage daily just before dusk. New World Monkeys It is generally agreed that if New World monkeys are not exposed to ultra- violet rays, vitamin D3 must be provided to prevent rickets and osteomalacia. Other nutritional requirements are similar to those for Old World monkeys. Old World Monkeys Almost all species of Old World monkeys can be adequately maintained on commercial diets. Supplementation-vitamins, minerals, and high-protein

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36 or high-caloric dietary preparations-may be indicated for infants, preg- nant animals, nursing mothers, and animals in an undernourished or other- wise poor condition. Presbytis entellus and P. cristatus are notable exceptions. These leaf- eating species require green alfalfa supplemented with fresh green vegetables. Without these nutrients they die of starvation. After 4 months in the labora- tory a conventional laboratory diet can replace the green vegetables, but the green alfalfa must be continued. Baboons Commercial rations adequately fortified with minerals and vitamins are usually suitable for routine colony maintenance. A formula with the follow- ing analysis has been found satisfactory: carbohydrate, 46.70 percent; fat, 9.35 percent; protein, 20.85 percent; CA/P, 1 to 1.2; calories per 100 g, 354. (See Table 1.) Chimpanzees Diets proposed for Old World monkeys (see above) are adequate for adult chimpanzees, provided they are given in appropriate quantities. Supplements of fruit and vegetables are desirable in order to relieve mo- notony in the diet. They may also be provided to correct a deficiency or for experimental purposes. See the bibliography for chimpanzee diet studies by Nees et al. EUTHANASIA Killing an experimental animal quickly and painlessly is an acceptable part of many investigations. Euthanasia may also be necessary to halt an ani- mal's suffering or to prevent a lingering death from disease. Euthanasia may be accomplished by administering an overdose of an injectable anesthetic agent, exposing the animal to an inhalant anesthetic for a prolonged period, or injecting one of the commercially available lethal compounds prepared for this purpose. A qualified individual should exam- ine the animal to assure that death has occurred before disposing of the carcass.

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37 TABLE 1 Recommended Raw-Ingredient Formula for Custom-Made Baboon Rations" Ingredient Percent Corn, yellow, ground 35 Wheat middlings, standard 20 Meat and bone meal 10 Soybean oil meal 10 Skim milk, dried 10 Alfalfa-leaf meal, dehydrated 2 Bone meal, steamed 2 Corn oil 6 Molasses, cane 2 Salt, iodized 1 Vitamin B compound premix* 1 812 5.1 mcg/lb Niacin 66.5 ppm Pantothenic acid 56.3 ppm Pyridoxine 15.0 ppmc Riboflavin 6.7 ppm Folic acid 2.2 ppm Thiamin 3.5 ppm Vitamin A stabilized concentrate, sufficient to furnish 10,000 IU per pound of ration Irradiated yeast, sufficient to furnish 700 ID of vitamin D per pound of ration) Ascorbic acid, 30 g per 100 pounds of ration Isonicotinic acid hydrazide, 0.015% "Recommended by the Southwest Foundation for Research and Education, San Antonio, Texas. &TotaI pyroxidine hydrochloride content to be not less than 1 g per 100 pounds of feed. C7.5 ppm without isoniazid. IDENTIFICATION AND RECORDS Identification of individual animals is generally desirable. Tattoos, neck chains, or collars may be used for this purpose. Records on experimental animals are essential to good animal care. No- tations concerning source, pertinent dates, vital statistics, health status, reproductive history, manipulations, and eventual disposition are recom- mended. Clinical records and necropsy reports should be accurately main- tained on each primate. tionVl