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Veterinary Care Veterinary care in laboratory animal facilities goes beyond the preven- tion, diagnosis, treatment, and control of disease. It also includes monitor- ing animal care and welfare and providing guidance to investigators on handling and immobilizing animals and preventing or reducing their pain and distress (NRC, 1985, 1992~. Responsibilities of the attending veterinar- ian are specified by the Animal Welfare Regulations (9 CFR 2.33, research facilities; 9 CFR 2.40, dealers and exhibitors). The first sections of this chapter deal with the procurement and condi- tioning of research dogs and the control of infectious and parasitic diseases. Aspects of veterinary care dealing with the use of anesthetics and analge- sics, surgery and postsurgical care, and euthanasia are taken up in the last three sections. The medical aspects of reproductive disorders are discussed in Chapter 4; special care for pups is also reviewed in Chapter 4 and ad- dressed in detail elsewhere (Hoskins, 19901. Reference values for blood analyses can be found in textbooks by Kaneko (1989) and Loeb and Quimby (1989~. Dogs can be afflicted with many uncommonly occurring but scientifi- cally interesting diseases and disorders, many of which also afflict humans. Some breeds have predispositions to particular diseases and disorders (e.g., dalmatians are prone to urate bladder stones); a comprehensive review of this subject is available (Willis, 1989~. Chapter 6 of this book addresses the maintenance of dogs with selected genetic disorders 51

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52 DOGS: LABORATORY ANIMAL MANAGEMENT PROCUREMENT General Considerations Dogs acquired from outside a research facility's breeding program must be obtained lawfully from dealers licensed by the U.S. Department of Agri- culture (USDA) or sources that the USDA has exempted from licensing (7 USC 2137~. A List of Licensed Dealers can be obtained from Regulatory Enforcement and Animal Care, Animal and Plant Health Inspection Service, USDA, Federal Building, Room 268, 6505 Belcrest Road, Hyattsville, MD 20782. Examples of exempt sources are municipal pounds and people who provide dogs without compensation. Procurement of dogs for research requires planning by a knowledgeable person to ensure that the dogs receive good care and that the needs of the investigator are met. The person should be familiar with federal regulations applicable to the acquisition of dogs (9 CFR, parts 2 and 3) and with state and local ordinances applicable to the aquisition of dogs from pounds and shelters. It is strongly recommended that institutions inspect vendors' pre- mises for compliance with procurement specifications agreed on by contract before the first dogs are purchased and periodically thereafter. Sources Both random-source and purpose-bred dogs can be purchased for re- search purposes. Random-source dogs are those raised under unknown conditions of breeding and health. Sometimes they are stabilized and con- ditioned (see below) by the dealer before sale. Purpose-bred dogs are those from known matings that have limited exposure to infectious diseases. Random-source dogs that have not been stabilized and conditioned by the vendor (often called nonconditioned random-source dogs) are usually acquired from USDA-licensed dealers or, less commonly, from pounds. If a number of dogs of similar weight or body conformation are needed, the purchaser must allow sufficient time for the group to be assembled by the vendor. Random-source dogs that have been stabilized and conditioned (often called conditioned random-source dogs) should be purchased only from vendors that have written standard procedures for their conditioning programs. Purpose-bred dogs are acquired from USDA-licensed dealers that breed dogs specifically for research or from an institutional breeding program. Dogs with diseases of research interest are often acquired from exempt sources, such as pet owners referred by clinical veterinarians.

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VETERINARY CARE 53 Conditioning Conditioning is defined as physiologic and behavioral adjustment to a new environment. The period required for that adjustment to occur is called the conditioning period. Conditioning consists of adjustment to a new regi- men, including new people, diet, climate, and exercise. The adjustment can be hastened if the using institution provides the same type of food as the dealer or vendor and uses the same type of automatic watering devices ~ 1~ _ _: _ 1 ~ 1 ~ . ~ ~ . r~ys~c status, as weft as tne presence of diseases, can be determined by assessing red-cell counts, packed-cell volumes, and white-cell differen- tial counts and by using blood urea nitrogen tests and other examinations of blood and urine. Those tests are most valuable when samples are taken several days after arrival, by which time initial adjustments to the new environment have been made. Abnormal findings on anv of the tests might warrant followup examinations. . A A _ ~ A. ~ ~ ~ ~ ~ ~ ~ J ~ ~v ~ v ~ ~ ~ ~ 45 , ~ . Evidence of behavioral adjustment includes decreases in fearful behav- ~ors, increases in friendly behaviors, increases in playfulness, and normal grooming behaviors. Some dogs might not adapt to human handling or the environment and are therefore inappropriate for use in long-term studies. The dealer should be questioned about the sources and histories of such dogs to determine whether additional dogs purchased from that dealer will be similarly distressed. Information on maladaptive canine behavior has been published elsewhere (Scott, 1970~. Many procedures such as trimming of nails, removal of matted fur, bathing, and teeth-cleaning can be performed during the conditioning pe- riod. There is no definitive rule about the optimal period for conditioning. The intended use of the dogs, the season, prevalence of canine diseases in the area, and other factors influence the length of the conditioning period. If the dogs are well selected, adequately socialized, immunized, and treated for parasites before delivery, the conditioning period can be reduced. Ran- dom-source dogs that have been held for 10 days or more by the dealer usually require at least 21 days of conditioning at the institution before one can be confident that they have adapted fully. Some prefer a minimal condi- tioning period of 45 days. The importance of humane treatment and proper care during conditioning must be emphasized. CONTROL OF INFECTIOUS DISEASES General Considerations There are three important strategies for controlling canine infectious diseases: examining dogs on arrival and refusing to accept dogs that exhibit

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54 DOGS: LABORATORY ANIMAL MANAGEMENT signs of disease, placing all newly acquired dogs in quarantine, and isolat- ing dogs that become sick. Some infectious pathogens to which dogs are susceptible could be introduced into an established colony by new arrivals, especially by random-source dogs, which are commonly unvaccinated. The most common of these pathogens are canine distemper virus (CDV); canine parvovirus (CPV-2~; canine herpesvirus (CHV); the respiratory agents ca- nine parainfluenza (PI-2), Bordetella bronchiseptica, and Mycoplasma spp.; canine adenovirus type 1 (CAV-1, infectious canine hepatitis) and type 2 (CAV-2, tracheobronchitis virus); and canine coronavirus (CCV). An addi- tional problem that warrants careful consideration is the possibility that unvaccinated random-source dogs can harbor rabies virus, which can have a long incubation period (Ache and Szyfres, 1987~. Dermatophytosis (ring- worm, principally Microsporum cants and M. gypseum) and canine papillomatosis (warts) can also present problems. Protection against these pathogens is discussed briefly below. Detailed information on canine infectious diseases is available in a number of general references (e.g., Appel, 1987; Barlough, 1988; Greene, 1990~. Quarantine Quarantine (in this context, the isolation of newly acquired animals until their health status has been evaluated) minimizes the risk of spreading diseases from newly arrived dogs to those already in the colony. In most facilities, the quarantine and conditioning periods overlap. During the quaran- tine period, most attention is directed to the control of infectious diseases and parasites. Procurement of dogs that are free of infectious diseases and parasites (i.e., conditioned random-source dogs or dogs bred specifically for research) reduces the time necessary for both quarantine and conditioning and might result in more reliable research results. Nonconditioned random- source dogs should be quarantined as a group, and no additional nonconditioned dogs should be introduced into the group. Quarantine facilities should be designed to provide physical barriers to the spread of infectious diseases (e.g., unidirectional airflow). That is espe- cially important when the research and quarantine facilities are parts of a single building. It is preferable for a quarantine facility to have its own animal-care technicians; however, if this is not possible, quarantined dogs should be cared for last. Newly arrived dogs should be housed singly to enable veterinarians and technicians to determine which dogs are not eating well, exhibit signs of disease, or are abnormal in other ways. Ideally, dogs are vaccinated by the dealer. If not, they should be vaccinated as soon as possible after arrival against CDV, CPV-2, and CAY-2 (such vaccination also protects against

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VETERINARY CARE 55 CAV-1~. Vaccination for PI-2 and B. bronchiseptica should be considered in institutions where respiratory disease is common. If the dogs are to be vaccinated against leptospirosis and rabies, that is usually done at the same time. If a person is bitten or scratched, the injured area should be cleansed, the person should be referred to appropriate medical personnel, and the dog should be isolated for at least 10 days, as recommended by the National Association of State Public Health Veterinarians (1993~. Research and Breeding Colonies The major threat to an established colony is that newly introduced dogs might harbor an infectious-disease agent or that personnel might carry such an agent into the colony on their hands or clothing. A regular immunization program, quarantine of nonconditioned random-source dogs, and rigorous sanitation practices will help to protect against infectious agents inadvert- ently introduced into an established colony. Annual vaccination with a multivalent vaccine is generally recommended, although immunity to CDV and CPV-2 generally persists for at least 3 years. In areas in which respira- tory disease is common, frequent vaccination (every 3 months) might be indicated. Frequent vaccination (every 6 months) with leptospira bacterins is recommended in areas in which leptospirosis is endemic or is a proven problem. Breeding Stock Some infectious diseases are of special concern in breeding colonies. CHV can remain undetected in a breeding kennel for years. When suscep- tible, pregnant (usually young) bitches are introduced into the colony, latent CHV manifests itself by causing abortions or fetal or neonatal deaths. A detailed discussion of CHV is available elsewhere (Carmichael and Greene, 1 990a). Canine brucellosis can severely affect reproduction in a breeding ken- nel. It is also a zoonosis. All dogs purchased for breeding stock should be tested for Brucella cants antibodies on arrival and placed in quarantine for at least 1 month, at which time a second brucellosis test should be run. New dogs should not be introduced into a breeding colony unless both tests are negative. An infected dog should not be used for breeding or for long- term studies. Beagles have an unusually high prevalence of brucellosis, although it is occasionally diagnosed in random-source dogs (Carmichael, 1979~. For detailed discussions of canine brucellosis see Carmichael (1990) and Carmichael and Greene (199Ob).

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56 Pups DOGS: LABORATORY ANIMAL MANAGEMENT CDV and CPV-2 infections are the principal viral diseases that threaten pups during the first 4 months of life, and prevention of these diseases should be the principal objective of an immunization program. Maternal antibody to CDV interferes with the development of an immune response to CDV vaccine; measles vaccine protects against disease but not infection in pups in which maternal antibody is still present (pups about 6-10 weeks old) (Baker, 1970~. CDV vaccine should be given to dogs by 14 weeks of age. No vaccines can prevent parvovirus infection in pups during one critical period-that during which they still have maternal antibodies that inhibit the response to vaccination but do not protect against virulent CPV- 2 (Carmichael, 19831. Proper management practices are critical in prevent- ing this infection. If a pup does contract the disease, it should be isolated immediately, and rigorous disinfection procedures should be implemented. Diseases caused by adenoviruses and CCV can occur in pups, but they are less common. It is generally recommended that modified live-virus vaccines-be used for immunization, if available. A killed-virus vaccine is used for rabies. A multivalent vaccine that protects against distemper, hepatitis, leptospirosis, and parvovirus and parainfluenza infections can be used. An intranasal vaccine against Bordetella bronchiseptica, which causes kennel cough, is generally recommended. Several vaccination regimens have been proposed (Baker et al., 1961; Carmichael, 1983; Swango, 1983~; one of them is given in Table 5.1 as a guide, but others are acceptable. The vaccination schedule should be adapted to address the perceived risk of infection. Pups can be vaccinated with intranasal vaccine against B. bronchiseptica at 3-4 weeks of age. Other than that, vaccinating pups less than 6 weeks old is not recommended, because vaccine safety has not been studied in very young pups. Isolation is more important than vaccination in prevent- ing disease in such pups. TABLE 5.1 A Vaccination Schedule for Pups Age Vaccine 6 weeks CDV or CDV combined with measles and CPV-2 8-10 weeks CPV-2 12-14 weeks Multivalent vaccine 16 weeks CPV-2 (or multivalent vaccine) and rabies

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VETERINARY CARE 57 Specific-Pathogen-Free Colonies Dogs from known matings that have never been exposed to specific infectious agents are called specific-pathogen-free (SPF) for those agents. These dogs are used in infectious-disease and vaccine-development research in which animals are required not only to be free from pathogenic agents, but never to have been exposed, either naturally or through vaccination, to pathogenic agents. It might also be preferable to use SPF dogs in some transplantation studies, because in profound immunosuppression, native and vaccinal viruses (e.g., CDV and CAY-1) might be activated and cause dis- ease (Thomas and Ferrebee, 1961~. The objective in preventing the outbreak of disease in SPF colonies is to isolate, rather than immunize, the dogs. Disease prevention depends on the establishment of physical barriers to preclude the introduction of dis- ease agents, rigorous management practices, and control of personnel move- ment into and within the facility (Sheffy et al., 1961~. Rodents and other pests that can transmit disease mechanically must be excluded. Purpose- bred SPF dogs are available commercially. If bred by the institution, initial breeding stock should be procured from dogs free of latent infectious agents, and all offspring taken by hysterectomy or cesarean section. Embryo-trans- fer technology offers additional possibilities for SPF colonies. Population immune status should be assessed periodically (at least once a year) by monitoring for antibodies to the common infectious diseases. In the event of an inadvertent infection that would compromise the use of the animals, the colony should be depopulated and re-established. CONTROL OF PARASITIC DISEASES Parasites are common in dogs, particularly random-source dogs. They can be found on the skin and hair and in the ears (ectoparasites) and in many internal organs, including the digestive tract, heart, lung, and blood vessels (endoparasites). Specific canine parasites are discussed briefly be- low; details on life cycles of, treatment for, and prevention or control of these parasites are found elsewhere (Georgi and Georgi, 1992~. Ectoparasites Ectoparasites include ticks, mites, lice, and fleas. Most can be easily eradicated with insecticides. Three ectoparasites commonly carried by ran- dom-source dogs can pose problems if they are not eliminated during quar- antine.

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58 DOGS: LABORATORY ANIMAL MANAGEMENT · The most damaging is probably the Rhipicephalus sanguineus tick. This tick can feed on dogs during all life-cycle stages, and once it enters a facility, it can be expensive to remove. · Mange caused by Sarcoptes scabei is sometimes inadvertently in- troduced into a facility on a dog that shows no overt signs of dermatosis. This parasite can be a particular problem in dogs that are group-housed or housed in cages or runs that allow the touching of body parts among ani- mals (e.g., through wire-mesh walls). Sarcoptic mange is treated by dip- ping the affected dogs and all dogs in contact with them in insecticide. It is probably also worthwhile to steam-clean enclosures and floors. · Fleas are commonly brought into facilities by random-source dogs. The flea life cycle can be disrupted by cleaning enclosures daily to remove developing eggs and larvae. Another strategy is to house dogs in enclosures raised more than 33 cm above the floor. Fleas cannot jump higher than 33 cm, so fleas that fall to or develop on the floor cannot reach the dog to feed. Additional ectoparasitic infestations that might persist in kennel set- tings include infestation with ear mites (Otodectes cynotis), "walking dan- druff" (Cheyletiella yasguri), and lice (Linognathus setosus, Trichodectes cants, and Heterodoxus spiniger). The canine nasal mite (Pneumonyssoides caninum) can also persist, but it is not known how often infestations with this mite occur in random-source dogs. To prevent the introduction of skin-dwelling ectoparasites, random-source dogs should be bathed or dipped before they are moved to the housing facility. Their ears should be examined and, if appropriate, treated for ear mites. Mites should be considered as the cause of persistent skin lesions, and appropriate action should be taken to make a correct diagnosis. All dogs, including random-source and breeding-colony dogs, are prob- ably host to the hair-follicle mite Demodex cants. Dogs probably become infected as puppies while nursing. Typically, the infestation is nonpathogenic; in rare instances, the mite causes severe mange. The development of demodectic mange in large numbers of kennel dogs is rare but has occurred. Treatment with topical applications or dips is possible as long as the lesions remain focal, but generalized demodectic mange often indicates some underlying problem (e.g., an inherited susceptibility to demodectic mange or a compro- mised immune system), and its treatment is difficult or impossible. En do parasites SPF dogs and purpose-bred dogs often host both protozoan and helmin- thic endoparasites. The protozoa include Isospora spp., Giardia spp., tri- chomonads, Cryptosporidium spp., Balantidium spp., and amebas. The hel- minths include ascarids (e.g., Toxocara cants and Toxascaris leonina), Filaroides

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VETERINARY CARE 59 spp., Strongyloides stercoralis, and occasionally hookworms and whipworms. If dogs are housed in a manner that allows mosquitoes access to them, they are also susceptible to infection with heartworm, Dirofilaria immitis. Isospora spp. have direct life cycles (i.e., no intermediate host is re- quired). Oocysts of these coccidia are commonly present in the feces of young dogs raised in colonies, and more than one species can be present in one dog. The oocysts of I. cants, I. ohioensis, I. neorivoltos, and I. burrows) are morphologically similar; however, those of I. cants are larger than those of the other three. Clinical signs include increased temperature and diar- rhea that is occasionally bloody. Infections usually subside after several days to weeks. Oocyst shedding decreases to low numbers 4 weeks after it begins. Chemoprophylaxis and basic sanitation are necessary to control the infection if it causes problems. Cryptosporidium is occasionally present in dogs in closed colonies, although it typically does not cause disease. In immunocompetent dogs, the small oocysts of Cryptosporidium are shed in low numbers, if at all, for a limited period; however, in immunosuppressed or immunocompromised dogs, Cryptosporidium can cause fatal disease. There is no proven method of chemoprophylaxis or treatment, but routine sanitation procedures, accompa- nied by regular steam cleaning of areas that might be contaminated, will . . . assist In reducing exposure to oocysts. Giardia cants is commonly present in both purpose-bred and SPF dogs. The prevalence is high in pups and decreases with age. The organism is spread between dogs by the fecal-oral transmission of resistant cysts. Typi- cally, pups are infected with Giardia and one or more species of Isospora; however, the infection usually causes little or no disease. As dogs mature, the number of organisms decreases. As with Isospora, chemoprophylaxis and basic sanitation are the most effective means of controlling Giardia. Trichomonas canistomae is a commensal organism present in the mouths of many dogs. It has no cyst stage and is transmitted between dogs by direct oral contact. There is usually no need for treatment. Species of Trichomonas and Pentatrichomonas are present in the large intestines of many laboratory-reared dogs. None of these species has a cyst stage; trans- mission is by the fecal-oral route. These organisms are sometimes observed in diarrheic feces in very large numbers, but they are usually not the cause of the diarrhea. Treatment is available but usually not necessary. Balantidium colt, a large ciliated parasite that is rarely found in dogs, and Entamoeba cold and E. histolytica, smaller ameboid parasites, are trans- mitted by cysts passed in the feces. These parasites are present in the large bowel. Their life cycles are similar to that of Giardia, and once they are established in a colony, they are easily perpetuated. The ascaridoid nematode (roundworm), Toxocara cants, is a common parasite of the small intestines of dogs, even in closed breeding colonies.

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60 DOGS: LABORATORY ANIMAL MANAGEMENT The parasite is transmitted from bitches to pups in utero, and pups begin to shed eggs in their feces a few weeks after birth. Once the eggs enter the environment, they require about 2 weeks to become infectious; they are very resistant to environmental extremes of heat, cold, and humidity. Pups should be treated soon after birth and several times during early life to prevent the development of adult roundworms from the stages obtained prenatally. Control measures should include steam cleaning of floors and disinfection of floors with a 1:4 (20 percent) solution of chlorine bleach. Adult dogs can have larvae in their tissues whether or not they are shedding eggs in their feces. It is possible to determine whether a dog has ever been infected by measuring antibody concentrations, and dogs that are Toxocara cants-naive are available commercially. The other canine ascaridoid, Toxascaris leonina, has a direct life cycle and does not infect pups transplacentally. The eggs of this parasite develop more rapidly than those of Toxocara cants but are just as resistant to ex- tremes of heat, cold, and humidity. Toxascaris leonina is commonly present in the small intestines of older purpose-bred and SPF dogs, but it is not known how the cycle is maintained in these colonies. Control and treat- ment are the same as those used for Toxocara cants. Filaroides hirthi is present in the lung parenchyma of many purpose- bred and SPF dogs. The lung lesions caused by the parasite can confuse histopathologic evaluations in toxicologic experiments. The life cycle is direct, and infective larvae are transmitted between dogs by oral or fecal- oral contact. Immunosuppressed dogs can become seriously ill as a result of auto-reinfection that leads to heavy parasite burdens. Infections can be treated, but control is difficult because fecal assays are insensitive. There- fore, all dogs in a contaminated room must be treated, not just those with positive fecal tests. Proper sanitation is helpful, but the larvae do not persist for long periods in the environment. Strongyloides stercoralis lives as a parthenogenetic female in the mu- cosa of the canine small intestine. Larvae develop to the infective stage 4-5 days after they are passed in feces. Transmission is by penetration of the skin by infective-stage larvae and by passage of tissue-dwelling larvae in the milk of lactating bitches. Immunosuppressed or immunocompromised dogs can develop severe disease as a result of auto-reinfection. S. stercoralis is also transmissible to humans. Although treatment is available, elimina- tion of the parasite from a breeding colony is difficult because it is not certain that transmammary transmission can be interrupted by chemothera- peutic measures. Routine removal of feces and cleaning of cage or pen floors reduce transmission. Adult hookworms live in the small intestine, where they cause blood loss and anemia. The hookworms Ancylostoma caninum and A. braziliense, like S. stercoralis, are transmitted through the milk or by larval penetration

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VETERINARY CARE 61 of the skin. However, infective-stage larvae are more likely to develop in soil than on a moist cage bottom fouled with feces, and transmission is more likely when dogs are housed outside on such surfaces as gravel or sand. Unlike dogs infected with S. stercoralis, dogs infected with hook- worms often show signs of overt disease, characterized by bloody diarrhea. In addition, hookworm eggs are much easier to detect in feces than are S. stercoralis larvae. Those differences and the dissimilarity of conditions required for larval development make it much less likely that hookworms will persist undetected in a colony. The hookworm Uncinaria stenocephala, which is present in more temperate climates, is transmitted mainly by larval ingestion; skin penetration and transmission in milk are uncommon. Thus, U. stenocephala is less likely to be perpetuated in a closed colony. Whipworms, Trichuris vulpis, live in the cecums and colons of dogs and cause large bowel disease that can produce bloody stools. The life cycle of this parasite is direct. Eggs are passed in feces and take several weeks to become infectious. They are highly resistant to environmental extremes, so contamination is very peristent if eggs get into the soil of earthen-floored runs. Dogs become infected by ingesting the infective eggs on soil-contaminated items. In the dog, the worms take about 3 months to develop to the adult stage, and reinfection is common. Treatment is avail- able but often has to be repeated. The filarioid nematode Dirofilaria immitis causes heartworm disease. It is transmitted between dogs by the bite of a mosquito. The prepatent period (the time between the inoculation of maturing forms by the mosquito and the first appearance of microfilariae in the host's blood) is slightly more than 6 months. The infection is often manifested as cardiopulmonary disease accompanied by respiratory distress and right-sided heart enlarge- ment. In dogs with patent disease, infections can be diagnosed by demon- strating microfilariae in the blood; however, some infected dogs do not have circulating microfilariae (Glickman et al., 1984.~. When it is impor- tant to ascertain that dogs are heartworm-free, serum or plasma can be examined with antigen-detection tests. Treatment for heartworm infection is generally precluded by its high cost, the stress it causes the dog, the length of time necessary for recovery, and the possibility of residual pathologic changes in the cardiovascular system. Where D. immitis is enzootic, dogs given access to outside runs should be protected by chemical prophylaxis. If dogs cannot be placed on chemi- cal prophylaxis, because of a study design or for other reasons, they can be protected by enclosing the outside kennels with screening. In addition to infection with the same parasites found in purpose-bred and SPF dogs, random-source dogs are likely to be infected with parasites that are relatively rare or that require intermediate hosts as part of their life cycles. If the intermediate hosts are uncommon (e.g., snails, then crayfish

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VETERINARY CARE TABLE 5.3 Signs, Degree, and Length of Surgically Produced Paina 65 Surgical Site Signs of Pain Degree of Pain Length of Pain Head, eye, ear, mouth Rectal area Bones Abdomen Thorax Spine, cervical Attempts to rub or scratch; self-mutilation; shaking; reluctance to eat, drink, or swallow; reluctance to move Rubbing, licking, biting, abnormal bowel movement or excretory behavior Reluctance to move, lameness, abnormal posture, guarding, licking, self-mutilation Abnormal posture (hunched), . , . anorexia, guarding Reluctance to move, respiratory changes (rapid, shallow), depression Abnormal posture of head and neck, reluctance to move, abnormal gait- "walking on eggs" Few signs, often moving Spine, thoracic or lumbar immediately Moderate to high Intermittent to continual Moderate to high Intermittent to continual Moderate to high: Intermittent upper part of axial skeleton (humerus, femur) especially painful Not obvious to Short moderate Sternal approach, Continual high; lateral approach, slight to moderate Moderate to severe Continual Slight Short Short aBased on observations of dogs. Reprinted from NRC, 1992. (a mixture of tiletamine and zolazepam). Each injectable drug has proper- ties that determine its duration of action and the route by which it is best administered. Ketamine is used as an anesthetic but its effectiveness as an analgesic for visceral pain is disputed (Booth, 1988b; Hughes and Lang, 1983~. It should be used in combination with another analgesic agent when visceral pain is expected. It can also induce seizure-like activity in dogs unless it is used in conjunction with another drug, such as diazepam, aceproma- zine, or xylazine. Chloralose and urethane are injectable anesthetics that have been used in some experiments; however, chloralose alone is a poor anesthetic that produces little analgesia unless it is combined with an opiate such as morphine (Rubal and Buchanan, 1986), or a short-acting anesthetic (Flecknell, 1987~. Urethane is mutagenic and carcinogenic (Auerbach, 1967; Mirvish, 1968~; it should be used with caution and only for nonsurvival surgery. Neuromuscular blocking agents (e.g., succinylcholine, atracurium, curare,

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66 DOGS: LABORATORY ANIMAL MANAGEMENT gallamine, pancuronium, and vecuronium) have no anesthetic or analgesic properties. They must not be used alone for surgical restraint, although they may be used in conjunction with anesthetic doses of general anesthetic drugs (NRC, 1985~. Local anesthetics (e.g., lidocaine, mepivacaine, and bupivacaine) act to disrupt nerve conduction temporarily. When applied around a nerve, they produce analgesia in the region served by that nerve. However, these drugs have no depressant effect on the brain; dogs undergoing procedures under local anesthesia usually must be restrained physically or chemically (e.g., with tranquilizers or sedatives). Specific techniques for regional anesthesia are described in several texts (Hall and Clarke, 1991; Lumb and Jones, 1984; Muir and Hubbell, 1989; Skarda, 1987; Soma, 19711. Local anesthet- ics alone are ordinarily used for only the most minor of surgical interven- tions; but they can be given either intrathecally or epidurally (usually via the lumbosacral space) to provide segmental anesthesia of caudal body parts sufficient for major surgery (e.g., celiotomy) (Skarda, 19871. Analgesics Opioid analgesics are compounds that act at specific opioid receptor sites in the central nervous system to produce analgesia. Table 5.4 lists some of these compounds. They are not general anesthetics, but can be used for surgery when combined with other appropriate drugs (NRC, 1992~. Opioid analgesics (e.g., oxymorphone) can be injected epidurally to control postsurgical pain for extended periods with minimal systemic effects (Popilskis et al., 1991~. Opioid agonists have been combined with tranquilizers to produce so- called neuroleptanalgesic combinations (e.g., a mixture of fentanyl and droperi- dol known by the trade name Innovar-Vet and produced by Pitman Moore, Mundelein, Ill.J. Such combinations are capable of producing a state that TABLE 5.4 Opioid Analgesics Used in Dogsa Drug Dose (mg/kg) Routeb Buprenorphine 0.01-0.2 IV, IM ButorphanoI 0.2-0.5 IV? IM Fentanyl 0.04 IV, IM Meperidine 2.0-6.0 IM Morphine 0.5-1.0 SC Oxymorphone 0.2-0.4 IV, IM aData from Harvey and Walberg, 1987. bIV = intravenous; IM = intramuscular; SC = subcutaneous

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VETERINARY CARE 67 sufficiently resembles general anesthesia to permit some surgical proce- dures (Muir and Hubbell, 1989; Soma and Shields, 1964~. Xylazine, which is classified as a sedative, has analgesic properties because of its action on central alpha-2 receptor sites. The nonsteroidal anti-inflammatory analgesics include acetaminophen, aspirin, flunixin, and ibuprofen. These drugs inhibit prostaglandin synthe- sis. They are ordinarily used to relieve the acute or chronic pain associated with inflammation and have little place in the management of severe or acute pain that is not associated with inflammation (NRC, 1992~. Recognition of Distress Not Induced by Pain Signs of distress caused by stressors other than pain include changes in behavior (e.g., unexpected aggression), maladaptive behaviors (e.g., stereotypies), and physical changes (e.g., weight loss). Experienced and attentive animal caretakers are of the utmost importance in early recognition of signs of distress. Changes in biochemical measurements (e.g., plasma cortisol con- centration) can also help in recognition of distress. Alleviation of Distress Not Induced by Pain Distress caused by stressors other than pain is often related to hus- bandry practices. Understanding and meeting dogs' social and physical needs will minimize or prevent such distress (NRC, 1992~. Phenothiazine tranquilizers, such as acepromazine (0.03-0.05 mg/kg in- travenously or intramuscularly, 1.0-3.0 mg/kg by mouth), are useful as preanesthetic drugs because they make unruly animals more tractable, re- duce the doses of anesthetic drugs necessary to maintain anesthesia, and make recovery from anesthesia smoother. However, they can have unpre- dictable effects and cause some animals to become excited rather than tran- quil (Voith, 19841. The phenothiazines have minimal antianxiety effects, and they are not the drugs of choice for decreasing fearful reactions (Marder, 19911. Alpha-2 agonists, such as xylazine (0.3-1.0 mg/kg intravenously, 0.5- 2.0 mg/kg intramuscularly), have many of the advantages of the phenothia- zines and are also good analgesics (Greed, 1987~. However, they can cause serious cardiovascular depression, hyperglycemia, and depressed thermoregula- tion, which can be reversed with yohimbine if necessary (Denhart, 1992~. Benzodiazepines, such as diazepam (0.1-0.5 mg/kg intravenously, 0.3- 0.5 mg/kg intramuscularly) are often used as adjuncts to injectable anes- thetic drugs, such as the barbiturates and ketamine, because they reduce the dose necessary to produce anesthesia and provide muscle relaxation (Greed, 1987!. Diazepam (Valium) is also used alone to treat seizures. Like the

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68 DOGS: LABORATORY ANIMAL MANAGEMENT phenothiazines, the benzodiazepines have an excitatory effect on some ani- mals. Because they are the drugs of choice for the treatment of fearful behaviors (Marder, 1991), especially fear of people (Hart, 1985), they can be useful in reducing distress in unsocialized dogs. However, the benzodi- azepines must be used with care in dogs that display fear-motivated aggres- sion. Decreasing the fear might make such dogs more likely to attack (Marder, 1991~. SURGERY AND POSTSURGICAL CARE Surgery in dogs should be performed in accordance with the tenets in the Guide (NRC, 1985~. The requirements for minor and nonsurvival surgi- cal procedures are less stringent than those for major survival surgical pro- cedures. Personnel performing surgical procedures must be adequately trained. Facilities for performing surgical procedures should be available as outlined in the Guide (NRC, 1985~. The successful practice of survival surgery requires strict adherence to aseptic surgical technique, as well as provision of adequate postoperative care and analgesia for the experimental subject. Aseptic techniques also have some value in major nonsurvival surgical pro- cedures (Slattum et al., 1991~. Generally, only healthy conditioned or pur- pose-bred dogs should be used for survival surgery. Familiarizing the dog with the laboratory environment can assist investigators in identifying in- tractable subjects and can be beneficial in decreasing postoperative stress. Presurgical Preparation Dogs should be surgically prepared by careful shaving to remove all hair from the surgical field. Shaving reduces contamination of the wound and avoids delays in healing that can occur if hair becomes matted in the incision. If a thermal cautery is to be used, an area should also be shaved for placement of a ground lead. Adherent grounding pads are available. The surgical field should be thoroughly cleaned with Betadine (povidone- iodine) or another appropriate surgical scrubbing material. Betadine sterile solution or other appropriate preparation should be applied to the entire field and allowed to dry. Underpadding used to absorb such solutions can be flammable and should be removed before surgery. All surgical instruments and chronic instrumentation must be sterilized with steam (autoclaving' or gas (ethylene oxide with proper poststerilization aeration time). Cold chemical sterilization is appropriate for minor surgical procedures, but exposure time must be adequate, and the instruments must be thoroughly rinsed in sterile saline before they come into contact with body tissues. All items should be packaged for sterilization in such a way

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VETERINARY CARE 69 that they can be opened and positioned for use without compromising steril- ity. Investigators should follow standard surgical practices: donning surgi- cal caps and masks, scrubbing, and donning surgical gowns and gloves. Sterile drapes should be positioned on the dog to define the surgical field. During the course of surgery, procedures for preserving sterility should be strictly followed. Generally, dogs should be treated with the appropriate preanesthetic medications (e.g., tranquilizers and atropine) to provide a degree of seda- tion and facilitate handling. General anesthesia is reviewed in the section "Alleviation of Pain" (see pages 64-67~; the type used depends on the type and duration of the surgical procedure. The adequacy of anesthesia can be assessed by the absence of the eyelid reflex and by the lack of withdrawal in response to painful stimuli (e.g., toe pinch). Insertion of a cuffed endot- racheal tube will ensure patency of the respiratory tract. The physiologic status of dogs under general anesthesia should be as- sessed by monitoring such parameters as pulse rate, systemic blood pres- sure, and respiratory rate. Electrocardiography can be used to monitor the status of the heart. A heating pad is useful for maintaining body tempera- ture. If inhalant anesthetics are used, the anesthetized dog should be venti- lated (tidal volume, 15-20 ml/kg; respiratory rate, 13-20 breaths/minute), and carbon dioxide should be monitored. Respiratory rate, tidal volume, and inspiratory-expiratory ratio can be adjusted to achieve acceptable end- tidal carbon dioxide (38-40 torr) and blood oxygen saturation greater than 90 percent. An intravenous catheter should be placed in the cephalic vein to pro- vide a continuous intravenous drip (e.g., of lactated Ringer's solution) for volume replacement and to ensure rapid access to the circulatory system. Depending on the situation, antibiotics can be administered through the catheter or intramuscularly. There is evidence that giving antibiotics during the 2 hours before surgery is more beneficial than giving them either during or after surgery (Classen, 1992J. -a r - - r Postsurgical Care Appropriate analgesics should be administered for postoperative pain, as needed (see pp. 66-67 and NRC, 1992~. Surgical wounds and sites of instrument entry into the body should be cleaned and treated daily (e.g., with 0.3 percent hydrogen peroxide or dilute Betadine solution). Topical antibiotics (e.g., bacitracin ointment) can be applied. should be changed every day. Basic biologic functions-including urination, defecation, and appetite- are good indicators of a dog's overall physical well-being. These are easy to observe and should be monitored regularly and often. Followup clinical -are Surgical dressings

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70 DOGS: LABORATORY ANIMAL MANAGEMENT examinations and laboratory tests can be used to identify specific problems. Appropriate supportive care should be provided as needed. A commonly used experimental protocol involving major survival sur- gery in the dog is the implantation of instruments that allow physiologic measurements over a long period while the dog is conscious. The dog is particularly suitable for this type of protocol because of its size, its equable temperament, and the close parallelism of its physiologic functions with those of humans. Strict adherence to the recommendations above will mini- mize confounding effects. EUTHANASIA Euthanasia is a method of killing an animal rapidly and painlessly (NRC, 1985~. It should be carried out by trained personnel following current guidelines established by the American Veterinary Medical Association (AVMA) Panel on Euthanasia (AVMA, 1993 et seq.; NRC, 1985) The method used must produce rapid unconsciousness and subsequent death without evidence of pain or distress, or the animal must be anesthetized before being killed (9 CFR 1.1~. The method used should also be safe for attending personnel, be easy to perform, and cause death without producing changes in tissues that might interfere with necropsy evaluation. Methods of euthanasia recom- mended by the AVMA Panel on Euthanasia (AVMA, 1993) are discussed below. Injection of Lethal Substances Injection of a lethal substance is probably the most suitable method for euthanatizing laboratory dogs. It usually involves the intravenous injection of a large dose of a barbiturate anesthetic, such as pentobarbital (more than 100 mg/kg). The advantage of this method is that the animal is anesthetized within seconds and does not undergo the pain or distress that might be associated with later respiratory and cardiac arrest. In fact, cardiac arrest can be delayed for many minutes after the onset of anesthesia; therefore, cardiotoxins (e.g., large doses of dibucaine) are sometimes used to hasten death (Wallach et al., 1981~. Unruly or aggressive dogs should be sedated or tranquilized to facilitate the restraint necessary for smooth intravenous injection. Intravenous injection is the preferred route of administration because venipuncture is easily performed on most dogs by trained, experi- enced personnel. Injection outside the circulatory system is less reliable, is potentially painful, and almost invariably produces a slow onset of action. Injectable drugs- such as magnesium sulfate, potassium chloride, and neuromuscular blocking agents (e.g., atracurium, curare, gallamine, pancuron- ium, succinylcholine, and vecuronium) may be used (Bowen et al., 1970;

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VETERINARY CARE 71 Hicks and Bailey, 1978~; however, the dogs must be in a deep plane of anesthesia before drug administration (AVMA, 1993~. Strychnine and nico- tine are not suitable for euthanasia, because their stimulant properties might cause distress even in anesthetized animals. Inhalation Methods Overdose of a potent inhalant anesthetic (e.g., halothane and isoflurane) is satisfactory for performing euthanasia on dogs and is particularly appro- priate for young dogs, in which venipuncture can be difficult. Anesthetic vapors tend to be irritating; therefore, the animals should be tranquilized first. If anesthetic vapors are used, a system for scavenging excess vapor is necessary to comply with federal guidelines on anesthetic-vapor pollution (CDC, 1977~. Ether, unlike most contemporary inhalant anesthetics, is flam- mable and explosive; therefore, its use is not recommended. Carbon monoxide and carbon dioxide both cause death by hypoxia. Carbon monoxide is impractical in most instances because of the risk to operators and the complexity of the equipment to administer it. Carbon dioxide has anesthetic properties and can be used for euthanasia (Carding, 1968; Leake and Waters, 1929~; however, unless the chamber is well de- signed and used properly, dogs can become distressed before becoming unconscious. Hypoxia is not satisfactory for euthanatizing pups because young animals tolerate hypoxia better than older dogs and can survive for more than 30 minutes (Glass et al., 1944~. Physical Methods Exsanguination is acceptable for euthanasia; however, the dog must be anesthetized because the decreasing blood flow causes anxiety and auto- nomic stimulation (Gregory and Wotton, 1984~. Electrocution is considered a humane method of euthanasia, provided that sufficient current passes through the animal's brain to produce unconsciousness before or coincidentally with the onset of cardiac arrest. However, this method of euthanasia is not practical in most laboratories because of the danger to personnel (AVMA, 1993; Roberts, 1954; Warrington, 1974~. Decapitation of pups is not rec- ommended by the AVMA Panel on Euthanasia (19931. Human Considerations Euthanasia of dogs or any other animals can be stressful for the person- nel performing the procedure. The degree of distress experienced by people observing or performing euthanasia depends on their backgrounds, personal philosophies, and ethical views on the use of animals in research (Arluke,

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72 DOGS: LABORATORY ANIMAL MANAGEMENT 1988~. People often transfer to the death of animals their unpleasant reac- tions to human death, and their responses to euthanasia can be magnified when strong bonds exist between them and the dogs being killed (e.g., strong bonds often develop between animal-care personnel and seriously ill canine models that require a great deal of care and rely totally on their human guardians). The stress experienced can be manifested as absentee- ism, belligerence, careless and callous handling of animals, and high turn- over rate. To be responsive to those concerns, institutional officials and supervisors should be aware of and sensitive to the issues and should pro- vide opportunities for individual and group discussion and support and for educational programs that furnish factual information about euthanasia and teach stress-management and coping skills (NRC, 1991~. REFERENCES Acha, P. N., and B. Szyfres. 1987. Rabies. Pp. 425-449 in Zoonoses and Communicable Diseases Common to Man and Animals, 2d ed. Scientific Pub. No. 503. Washington, D.C.: Pan American Health Organization. Amyx, H. L. 1987. Control of animal pain and distress in antibody production and infectious disease studies. J. Am. Vet. Med. Assoc. 191: 1287- 1289. Appel, M. J., ed. 1987. Virus Infections of Carnivores. Amsterdam: Elsevier Science Publishers. 500 pp. Arluke, A. B. 1988. Sacrificial symbolism in animal experimentation. Object or Pet? Anthrozoos 2(2):98-117. Auerbach, C. 1967. The chemical production of mutations. Science 158:1141-1147. AVMA (American Veterinary Medical Association). 1993. 1993 Report of the AVMA Panel on Euthanasia. J. Am. Vet. Med. Assoc. 202:229-249. Baker, J. A. 1970. Measles vaccine for protection of dogs against canine distemper. J. Am. Vet. Med. Assoc. 156:1743-1746. Baker, J. A., D. S. Robson, L. E. Carmichael, J. H. Gillespie, and B. Hildreth. 1961. Control procedures for infectious diseases of dogs. Proc. Anim. Care Panel 11:234-244. Barlough, J. E., ed. 1988. Manual of Small Animal Infectious Diseases. New York: Churchill Livingstone. 444 pp. Booth, N. H. 1988a. Section 4: Drugs acting on the central nervous system. Pp. 153-405 in Veterinary Pharmacology and Therapeutics, 6th ea., N. H. Booth and L. E. McDonald, eds. Ames: Iowa State University Press. Booth, N. H. 1988b. Intravenous and other parenteral anesthetics. Pp. 212-274 in Veterinary Pharmacology and Therapeutics, 6th ea., N. H. Booth and L. E. McDonald, eds. Ames: Iowa State University Press. Bowen, J. M., D. M. Blackmon, and J. E. Haevner. 1970. Effect of magnesium ions on neuromuscular transmission in the horse, steer, and dog. J. Am. Vet. Med. Assoc. 157:164- 173. Carding, A. H. 1968. Mass euthanasia of dogs with carbon monoxide and/or carbon dioxide; preliminary trials. J. Small Anim. Pract. 9:245-259. Carmichael, L. E. 1979. Brucellosis (Brucella canis). Pp. 185-194 in CRC Handbook Series in Zoonoses, vol. 1, J. H. Steele, ed. Boca Raton, Fla.: CRC Press. Carmichael, L. E. 1983. Immunization strategies in puppies why failures? Compend. Contin. Educ. Practicing Vet. 5: 1043- 1051.

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VETERINARY CARE 73 Carmichael, L. E. 1990. Brucella cants. Pp. 335-350 in Animal Brucellosis, K. Nielsen and J. R. Duncan, eds. Boca Raton, Fla.: CRC Press. Carmichael, L. E., and C. F. Greene. 1990a. Canine herpesvirus infection. Pp. 252-258 in Infectious Diseases of the Dog and Cat, C. E. Greene, ed. Philadelphia: W. B. Saunders. Carmichael, L. E., and C. E. Greene. 1990b. Canine brucellosis. Pp. 573-584 in Infectious Diseases of the Dog and Cat, C. E. Greene, ed. Philadelphia: W. B. Saunders. CDC (Centers for Disease Control). 1977. Criteria for a Recommended Standard Occupa- tional Exposure to Waste Anesthetic Gases and Vapors. HEW Pub. No. NIOSH 77-140. Washington, D.C.: U.S. Department of Health, Education, and Welfare. 194 pp. Avail- able by interlibrary loan from the CDC Information Center, M/S C04, Atlanta, GA 30333. Classen, D. C., R. S. Evans, S. L. Pestotnik, S. D. Horn, R. L. Menlove, and J. P. Burke. 1992. The timing of prophylactic administration of antibiotics and the risk of surgical-wound infection. N. Eng. J. Med. 326:281-286. Denhart, J. W. 1992. Xylazine reversal with yohimbine. Pp. 194-197 in Current Veterinary Therapy. XI. Small Animal Practice, R. W. Kirk and J. D. Bonagura, eds. Philadelphia: W. B. Saunders. Flecknell, P. A. 1987. Special techniques. Pp. 59-74 in Laboratory Animal Anaesthesia. An Introduction for Research Workers and Technicians. London: Academic Press. Georgi, J. R., and M. E. Georgi. 1992. Canine Clinical Parasitology. Philadelphia: Lea & Febiger. 227 pp. Glass, H. G., F. F. Snyder, and E. Webster. 1944. The rate of decline in resistance to anoxia of rabbits, dogs and guinea pigs from the onset of viability to adult life. Am. J. Physiol. 140:609-615. Gleed, R. D. 1987. Tranquilizers and sedatives. Pp. 16-27 in Principles & Practice of Veterinary Anesthesia, C. E. Short, ed. Baltimore: Williams & Wilkins. Glickman, L. T., R. B. Grieve, E. B. Breitschwerdt, M. Mika-Grieve, G. J. Patronek, L. M. Domanski, C. R. Root, and J. B. Malone. 1984. Serologic pattern of canine heartworm (Dirofilaria immitis) infection. Am. J. Vet. Res. 45:1178-1183. Greene, C. E., ed. 1990. Infectious Diseases of the Dog and Cat. Philadelphia: W. B. Saunders. 971 pp. Gregory, N. G., and S. B. Wotton. 1984. Time to loss of brain responsiveness following exsanguination in calves. Res. Vet. Sci. 37:141-143. Hall, L. W., and K. W. Clarke. 1991. Veterinary Anaesthesia, 9th ed. London: Bailliere Tindall. 410 pp. Hart, B. L. 1985. Behavioral indications for phenothiazine and benzodiazepine tranquilizers in dogs. J. Am. Vet. Med. Assoc. 186:1192-1194. Harvey, R. C., and J. Walberg. 1987. Special considerations for anesthesia and analgesia in research animals. Pp. 380-392 in Principles & Practice of Veterinary Anesthesia, C. E. Short, ed. Baltimore: Williams & Wilkins. Hicks, T., and E. M. Bailey, Jr. 1978. Succinylcholine chloride as a euthanatizing agent in dogs. Am. J. Vet. Res. 39:1195-1197. Hoskins, J. D. 1990. Veterinary Pediatrics: Dogs and Cats from Birth to Six Months. Phila- delphia: W. B. Saunders. 556 pp. Hughes, H. C., and C. M. Lang. 1983. Control of pain in dogs and cats. Pp. 207-216 in Animal Pain: Perception and Alleviation, R. L. Kitchell and H. H. Erickson, eds. Bethesda, Md.: American Physiological Society. Kaneko, J. J., ed. 1989. Clinical Biochemistry of Domestic Animals, 4th ed. San Diego: Academic Press. 932 pp. Leake, C. D., and R. M. Waters. 1929. The anesthetic-properties of-carbon dioxide. Curr. Res. Anesth. Analg. 8:17-19.

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VETERINARY CARE 75 animals. Pp. 227-234 in Nutrition and Behaviour in Dogs and Cats, R. S. Anderson, ed. Oxford: Pergamon Press. Wallach, M. B., K. E. Peterson, and R. K. Richards. 1981. Electrophysiologic studies of a combination of secobarbital and dibucaine for euthanasia of dogs. Am. J. Vet. Res. 42:850-853. Warrington, R. 1974. Electrical stunning, a review of the literature. Vet. Bull. 44:617-628. Willis, M. B. 1989. Genetics of the Dog. London: H. F. & G Witherby. 417 pp.