Use of Random Source Dogs and Cats for Research
The statement of task given to this Committee by NIH was specific to the use of animals from Class B dealers in scientific research, but this report addresses the use of random source dogs and cats in particular, per the intent of Congress (as discussed in the Summary and Chapter 1). The two types of animals, random source and Class B, are inextricably linked but also differ; Class B dealers acquire both random source and non–random source animals, as defined in Chapter 1. As detailed in Chapter 4, only 20 percent of dogs from Class B dealers are clearly identified as random source animals from pounds and shelters. Thus, most dogs from Class B dealers are non–random source and similar to those available through other sources. Because random source animals and specifically random source animals from pounds and shelters are the driving force for Congressional and public concern, and are the animals of interest to NIH, the Committee was compelled to discuss the specific attributes, both desirable and undesirable, of random source animals in this report.
Dogs and cats, regardless of source, have been used in American biomedical research for over a century, and random source dogs and cats have contributed to advances in both human and animal health. But the American public is divided in its opinions about the use of dogs and cats from shelters and pounds in research. Public attitudes are difficult to measure accurately, however, since opinion polls are often biased to serve the needs or perspective of the polling agency. For example, in a 1990s public opinion poll conducted by the Survey Research Center of the University of Michigan Institute for Social Research, 61 percent of respondents favored the use of unwanted animals from the pound for medical research and only
23 percent were against such use. Similarly, 75 percent would oppose a law to prevent unclaimed pound animals from being used in medical research for the public benefit (Michigan Society for Medical Research [MISMR]).1 But the results of the Michigan poll must be balanced with the knowledge that it is a regional poll, limited in scope (see Box 2-1).
At the other end of the spectrum, the results of a national poll conducted by the American Humane Association in 1988 showed that many members of the public opposed pound seizure (discussed further in Chapter 4) because they viewed shelters as havens for homeless animals and not a resource for biomedical research (American Humane Shoptalk 1988). This perspective is shared by some academic institutions, exemplified by the Colorado State University College of Veterinary Medicine and Biological Sciences (CVMBS) policy on animal use: “College policy prohibits the acquisition of live animals from shelters, either directly or indirectly through third party vendors, for use in research or teaching. The College recognizes that many individuals in our society are opposed, on ethical and scientific grounds, to the release of animals from shelters (pound seizure) for use in research or teaching. This objection is founded in the understanding that pounds or animal shelters were not designed as facilities to supply animals for such activities. Rather, they were developed to be places where people may bring unwanted or stray animals in the hope of a new home being found. If not successfully adopted, the animals may be euthanized. The release of these animals for research or teaching may be interpreted as a breach of the public trust that could lead to loss of public support” (CVMBS 2006a). In addition to concern about the use of pound animals in research, the CVMBS policy also addresses the quality of care provided to the animals used by the College: “In selecting sources from which to purchase animals to be used in research and teaching, the CVMBS strives to patronize only those suppliers who maintain the highest standards of animal care. Examples of preferred animal sources for teaching and research include: Animals typically available through well-established, federally licensed and regulated sources of purpose-bred and raised animals for teaching and research are used exclusively for species such as dogs and cats” (CVMBS 2006b).
The tendency to view dogs and cats as family members has become stronger in the past 20 years, as evidenced not only by polls (according to a 2007 Harris poll, 88 percent of pet owners view their pets as family members) but also by increased spending on veterinary care, food, toys, clothing, and day care, and by the PETS Act passed by Congress in 2006 (Harris Poll 2007). After Hurricane Katrina, when scores of people either refused to evacuate and/or returned home early out of concern for their pets,
the PETS Act mandated that disaster plans include provisions for companion animals (The White House 2006). The public has also become increasingly vocal in support of improved care for pound animals and in opposition to the euthanasia of adoptable shelter animals, as evidenced by the rise in the number of “no kill” pounds and shelters and by veterinary specialization in shelter medicine (Zawistowski 2008). It is unlikely that public opinion has shifted dramatically to now favor pound seizure.
The professional and scientific communities view the issue somewhat differently. The American Veterinary Medical Association, in its November 2007 official policy position statement, “believes there is ample justification for prudent and humane use of random source dogs and cats in research, testing and education.”2 The American Physiological Society (APS) supports the continued use of random source animals, recognizing that they have attributes that are important in the fields of study relevant to its members: “The American Physiological Society recognizes the importance of research that depends upon animals of large size, advanced age, and diverse genetic background. These are known as ‘random source animals’…”3
THE “3RS” PRINCIPLE
The universal principle that guides biomedical research on animals is the “3Rs” doctrine of Russell and Burch (1959; see also NRC 2003) that promotes reduction, refinement, and replacement of research animals whenever scientifically feasible. As discussed in Chapter 2, the number of dogs and cats used in research has been dwindling for the past 20 years, and random source dogs and cats make up a very small percentage of those animals. Although many animals in shelters and pounds are elderly or terminally ill and brought to shelters by their owners for immediate euthanasia (Kass et al. 2001), substantial numbers are otherwise healthy and could in theory be used for biomedical research studies. In addition, if these animals are not accessible for research, additional purpose-bred animals must be generated to fill the need. Therefore, some might argue that failure to use unwanted pound and shelter animals for research runs counter to the “reduction” component of the 3Rs principle. In contrast, others would argue that use of random source animals does not address the “refinement” or “replacement” components, or the “reduction” of the overall number of animals used. Thus, even this issue is not straightforward.
DESIRABILITY OF RANDOM SOURCE DOGS AND CATS FOR RESEARCH
One of the challenges of animal-based research is identification of an optimal model for biomedical research endeavors. Well-chosen animal models provide reproducible insight into normal function, disease states, and effectiveness of drugs and devices for treatment. Animal models that are less than optimal decrease the quality of knowledge and increase the chance for adverse drug and device events. As a result, the search for the best animal model is essential for understanding diseases and developing treatments for them.
Random source dogs and cats represent a potentially important source of animals with unique anatomic and physiologic attributes as well as naturally occurring diseases such as cancer, genetic diseases, age-related diseases, and infectious diseases. The Committee emphasizes that its task was to identify “common research topics” for which these animals are “desirable” and to describe “specific characteristics” that make them “particularly well suited” for these studies. The Committee was not tasked with comparing attributes of random source animals to those of purpose-bred animals nor with identifying attributes unique to random source or Class B dogs and cats.
The supposedly greater tractability of random source dogs and cats is sometimes cited as an advantage for their use. For example, opinion provided to the Committee by some investigators through the APS (personal communication to the Committee from David Kass, October 2008) indicated that random source animals were often behaviorally more predisposed than purpose-bred animals to training such as resting quietly for conscious animal studies or running on a treadmill. While tractability is certainly an important trait for studies requiring measurement of blood pressure, heart rate, and circulating hormones in conscious animal models, it is important to emphasize that this trait is largely a function of prior socialization with humans and therefore not confined to random source animals. Poorly socialized dogs and cats, regardless of source, can be expected to be more fearful of, and resistant to, interactions with unfamiliar people including laboratory personnel (Serpell and Jagoe 1995; Turner 2000). Conversely, properly socialized purpose-bred animals can be as tractable as former pets. Therefore, generalizations regarding tractability cannot be made, and depend on individual animals and their socialization and history.
Furthermore, according to the AWA, PHS Policy, and the Guide, justification for the use of a particular species is required for approval of a scientific protocol, but justification of the source of such animals is not. Because there is no regulatory requirement to maintain records of the source(s) of research animals, documentation and justification for the use of dogs and
cats from random sources (such as Class B dealers, pounds, and shelters) are not available. Given this lack of information, the “necessity” of the use of these animals is nearly impossible to determine. Nonetheless, the Committee was able to identify fields (described in the next section) and “common research topics” where the potential exists to use random source animals, including in NIH-funded research, and describe the particular characteristics that may make these animals well suited for research in these areas. It is important to emphasize that these characteristics may not be unique to random source animals and that in many cases other animals, including Class A animals, may also have these particular characteristics.
RANDOM SOURCE DOGS: ANATOMIC AND PHYSIOLOGIC ATTRIBUTES
Scientific investigation may require the use of older, larger, or genetically diverse dogs, or dogs with naturally occurring disease, any of which may be available as random source animals. In contrast, purpose-bred dogs, such as those supplied by Class A dealers, tend to be young and healthy; they include beagles, “mini-mongrels,” and hounds weighing 23-27 kg (50-60 pounds) with a defined genetic background and disease-free status suitable for many types of biomedical research.
A common argument for the use of random source dogs is the need for larger (27-37 kg, or 60-80 pounds) and older animals that are physically and physiologically similar to humans (Parsons et al. 1996; Sasajima et al. 1999). But demand for these larger and older animals is usually not great and maintaining even small numbers of larger animals for long periods may not be cost effective for vendors of purpose-bred dogs.
Large mixed-breed random source dogs have been used in the study of cardiac diseases, and in the development of procedures and devices to alleviate them, because of their size, depth of the chest cavity, and large heart and great vessels (aorta and pulmonary arteries). These features allow adequate working space to perform complex cardiac procedures and accommodate human commercially produced devices for testing.
The dog’s cardiovascular system is similar to that of humans in both size and function. Anatomically, the dog’s coronary artery system mimics chronic remodeling in humans following myocardial ischemia with extensive subepicardial collateral vessels and can serve as a model for regional and global myocardial ischemia (Swindle and Adams 1988). But there are differences in coronary artery anatomy and cardiac physiology between random source dogs and purpose-bred dogs, and these differences (or “con-
ditioning”) can affect the animal’s physiological status. Data presented on behalf of the American Physiological Society indicated that random source animals exhibited a greater increase in coronary blood flow and myocardial oxygen consumption (Tune et al. 2000; personal communication, Bill Yates, to Committee, October 2008). Furthermore, the incidence of idiopathic extramural coronary arteritis occurred less often in purpose-bred animals than in random source animals (Hartman 1989).
The dog’s coronary sinus, or venous drainage of the heart, is also similar to human anatomy, allowing for investigation of chronic resynchronization therapy and development of devices and procedures to treat severe congestive heart failure (Reising et al. 1998; Williams et al. 1994). Physiologically, the cardiac electrical conduction system in the dog mimics that of humans, so dogs are used for studies of normal and abnormal cardiac conduction, including atrial fibrillation and other dysrhythmias (Lee et al. 2006).
Random source animals have also been used to study dilatative cardiomyopathy using an induced rapid pacing model. These dogs had cardiac myosin isoform shifts (myosin heavy chain (MHC)-b and ventricular light chain (VLC)-2) in the heart chambers similar to those observed in end-stage human heart failure (Fuller et al. 2007). Conditions have been identified in random source animals that specifically contributed to identification and treatment of mechanisms associated with cardiac arrhythmias—including Long QT syndrome, Brugada syndrome, and Timothy’s syndrome—that are not present in purpose-bred dogs. For example, when purpose-bred beagles were used for research associated with Brugada syndrome, they were found to be unsuitable due to the lack of certain ion channel mutations, whereas random source dogs developed the characteristics of this arrhythmia (Antzelevitch 2008).
Scientists investigating diseases in pulmonary medicine and using thoracic surgical procedures seek barrel-chested large breed dogs for several reasons. Pulmonary function studies use dog models because of physiologic aspects such as increasing microvascular pressure creating pulmonary edema (Swindle and Adams 1988), which has been used as a model for acute respiratory distress syndrome (Reising et al. 1998) and acute lung injury (Kaczka et al. 2005) in humans. Large dogs have a readily accessible single pulmonary artery and vein of the left lower lung lobes, allowing for ease of cannulation and analysis of pulmonary metabolism. Historically, lung transplant procedures were developed using large random source dogs because of the deep chest cavity, again allowing access for complex anastomoses of vascular and airway structures (Blumenstock et al. 1981).
Random source dogs have been and continue to be integral to the development of prosthetic devices for hip and knee replacements and of fixation devices and techniques, as well as vertebral fusion models, tendon and ligament repair, and assessment of biomaterials for orthopedic procedures (Arnoczky et al. 1982; Greis 2001). In some circumstances, the larger animal’s size accommodates human prosthetic devices, but many of these materials and devices eventually are designed for veterinary use in smaller animals. Thus medical advances with research dogs now afford companion dogs many of the same benefits as for humans, such as hip and knee replacement, arthroscopic ligament repair, meniscectomy, and other procedures associated with degenerative joint disease.
Older dogs have been used to study osteoarthritis, cervical disc degeneration, and vertebral fusion because the pathophysiology of the mature articular surfaces and vertebral disc is similar to that of aged humans (An and Friedman 1999; Hunter et al. 2004; Smith et al. 1998). Cervical disc degeneration occurs naturally in older large breed dogs and the cervical and lumbar disc spaces are large enough to support artificial disc prosthetics and materials used for fusion or replacement of this structure (Cook et al. 1994). Many orthopedic studies use older, skeletally mature animals to reflect an adult human population rather than younger (less than 1-year-old) dogs (Frick et al. 1994). In humans, intervertebral disc disease is preceded by the disappearance of notochordal cells in the nucleus pulposus (inner portion of the disc). Similarly, older (5-year-old) mixed-breed dogs have few notochordal cells in the nucleus pulposus and are considered to be an adequate model of the human clinical condition (Hunter et al. 2004). Therefore, older large breed random source dogs have been used and are desirable for these studies (Hasegawa et al. 1995; Katsuura and Hukuda 1994; Nguyen-minh et al. 1997).
Rodent and primate studies indicate that older animals are physiologically different from younger animals (Ferrari et al. 2003). Advanced age is an attribute commonly found in random source animals and may make them desirable for research.
Random source dogs may have age-related chronic or persistent disease conditions such as congestive heart failure, arthritis, allergy, dementia, and neoplastic conditions that may make them desirable for investigations into similar human conditions. For example, canine osteosarcoma has a predictable metastatic rate and pattern that make it attractive for studies of antimetastatic approaches. Canine and feline malignant mammary tumors
have a similar metastatic pattern to that of mammary tumors in women, namely metastasis to the regional lymph nodes and lung (MacEwen 1990). More recently, random source animals have been used in NIH-funded studies of the ocular system, dementia, and cardiac function (Anyukhovsky et al. 2005; Dun et al. 2003; Goralska et al. 2007, 2009; Studzinski et al. 2006; Taylor et al. 2004).
Advanced age itself, independent of disease conditions, may be desirable for some studies. Several studies investigating veterinary and human pharmaceuticals have revealed varying efficacies and toxicological side effects related to the age of the animal subjects. For example, a COX-2 inhibitor intended to treat older, arthritic animals was recently developed and toxicologically tested using only young beagle dogs. Once on the market, it was discovered that older dogs metabolized the drug very differently, resulting in severe side effects that included gastric ulcers, liver and kidney damage, and death.4,5
Acquisition of aged dogs poses a logistical and financial challenge that can be addressed with random source animals. Representatives of one purpose-bred vendor testified that they could provide older animals (retired breeders) on a limited basis but that they are unavailable in substantial numbers; purpose-bred animals generally are sold as young as possible (usually 6-9 months) to minimize the expense of housing (personal communication with Class A vendors). In addition, the average duration of NIH grants usually prohibits an investigator from requesting animals years before they are required given the lack of certainty of funding beyond a single grant cycle. It would be reasonable to assume that the cost of maintaining dogs and cats for several years would be passed on to the users (personal communication with Class A vendors), as vendors of purpose-bred animals would be unlikely to sustain the costs of maintaining the animals for a long time unless they knew a customer base was available to purchase them at or beyond a certain age.
The challenge of funding is illustrated by an example of recent work on a canine model of dementia in the aged beagle. Approximately 20 animals from a single colony were used for these studies over a 2- to 3-year period. The multicenter investigative team was supported by up to four NIH individual investigator grants and by several other significant non-NIH sources, all of which represent a level of combined extramural support far beyond that typically attained by individual NIH-funded investigators (Opii et al. 2008; Siwak-Tapp et al. 2007, 2008). On the other hand, this work also exemplifies an alternative for access to aged animals through existing purpose-bred research colonies.
Genetic diversity may be an attribute necessary for some aspects of current and future biomedical research, and the genetic diversity represented among the many breeds in the general dog population cannot be reproduced in purpose-bred colonies. Furthermore, maintenance of maximal genetic diversity in a single colony of dogs would require more than 200 breeding pairs (personal communication from Stephen O’Brien to the Committee, January 2009). Nobel laureate Dr. E. Donnall Thomas, who received the award for his work in bone marrow transplantation, stated that “marrow grafting could not have reached a clinical application” (Thomas 1990, pp. 581-582) without the use of outbred dogs. Non-purpose-bred dogs have also been critical in the development of hematopoietic cell transplantation or bone marrow transplantation because of their genetic diversity, large size, long life, and the fact that, other than humans, they are the only mammals to possess these qualities (Ostrander and Wayne 2005). In addition, genetically diverse animals have also been instrumental in studies of total body irradiation, chemical and radioimmunological myeloablation, in vivo and in vitro graft manipulation, and graft-versus-host disease studies (Lupo and Storb 2007).
Naturally Occurring Infectious Diseases
Random source dogs exposed to outdoor environments and various vectors that may carry disease can be effective models of naturally occurring infectious diseases. Vector-borne diseases such as heartworm (Dirofilaria immitus), Lyme disease (Borrelia burgdorferi), Rocky Mountain spotted fever (Rickettsia rickettsii), babesiosis (Babesia microti), ehrlichiosis (Ehrlichia canis), and/or the antibodies to these organisms can be identified in random source dogs that have been exposed to outdoor environments (Scorpio et al. 2008). Random source animals may also have Sarcoptic mange (Sarcoptes scabiei), Demodectic mange (Demodex canis), or coccidiosis from natural exposure to parasites. To maintain the naturally occurring infection, standard conditioning or treatments for these parasitic diseases may be withheld from some random source animals so that they are available for studies involving these infections.
Research on naturally occurring infectious diseases of dogs is generally not supported by the NIH, but some members of the Committee believed that it was important to point out that the U.S. Department of Health and Human Services Food and Drug Administration (FDA) Center for Veterinary Medicine’s (CVM) Guidance Document for New Animal Drug Applications6
(Guidance 61) states that for dose determination studies natural infections are ideal, whereas induced infections are acceptable. In addition, Guidance 90 “Guidance for Industry – Effectiveness of Anthelminthics: General Recommendations, Final Guidance” states that the use of natural or induced infections in effectiveness studies should be determined by the type of parasite and the claim proposed by the sponsor. Finally, according to the International Harmonization of Anthelminthic Efficacy Guidelines7 (VICH GL#19, FDA/CVM Guidance #111), “Dose confirmation studies should be conducted using naturally or artificially infected animals; however, at least one study should be conducted in naturally infected animals for each parasite claimed on the label.” Therefore, although studies on naturally infected dogs do not typically apply to NIH-funded research, random source animals may be important for other types of research.
Spontaneously Occurring Animal Models of Human Disease
The genetically diverse pet population has been the source of unique animal models that are not available from vendors of purpose-bred animals; for example, diseases have been identified in a mixed population of pet animals in Germany (Neumann and Bilzer 2005), and random source animals have served as controls for studies in comparison to purebred animals (Basso et al. 2004; Smucker et al. 1990).
Most often, spontaneously occurring diseases have been identified in a particular breed and a colony established using non-purpose-bred animals. For example, spontaneous genetic animal models for sleep apnea, muscular dystrophy, progressive retinal atrophy, hereditary nephropathy, and hemophilia A and B have been identified in non-purpose-bred dogs (Canine Inherited Disorders Database;8 Wolfe 2009). There are no other large animal models for these diseases. In some circumstances, individual investigators have established breeding colonies to study these diseases. Examples of dog colonies maintained at research facilities as models of genetic disease include hemophilia A dogs derived from Irish setters, hemophilia B dogs derived from Lhasa Apsos, von Willebrand disease dogs derived from Scottish terriers, and Duchenne muscular dystrophy dogs derived from golden retrievers (Nichols et al. 2009; Wang et al. 2009).
In addition, other genetic diseases have been identified in breeds of dogs used for gene-based therapy. The Swedish Briard (RPE65) is the only dog
breed that has responded successfully to gene therapy for retinal degeneration, opening the door for several human clinical trials. Alaskan Malamutes and German shorthaired pointers may also provide similar success in gene therapy for achromatopsia (Stieger et al. 2009). Finally, naturally occurring dog and cat models for human genetic heart diseases exist and are critical for the development of gene-based therapy; for example, Portuguese water dogs are maintained at the University of Pennsylvania as a model for dilatative cardiomyopathy (Sleeper et al. 2009).
These valuable models are examples of the desirability or necessity of access to random source animals as genetically diverse control animals, and of as yet undetermined animal models that may result from naturally occurring single nucleotide polymorphisms, epigenetic occurrences, or other genetic alterations (personal communication, Stephen O’Brien to the Committee, January 2009). Discovery of new models of human disease has not typically arisen through large-scale random screening of random source dogs from shelters, pounds, or Class B dealers. Instead, these animals are usually sought out as naturally occurring disease models based on knowledge of their availability from random sources. The development of novel dog models of human disease relies on a sophisticated process of referral by breeders or veterinarians aware of nuances in a certain breed, veterinary medical workup, scientific characterization, and validation as an animal model. Such programs are ongoing with NIH support for the discovery of novel models in dogs and cats (see Chapter 4).
RANDOM SOURCE CATS: ANATOMIC AND PHYSIOLOGIC ATTRIBUTES
Cats have long been a mainstay of NIH-funded studies of neurological, cardiovascular, and respiratory diseases and the immune system. The similarity of these physiological systems to those of humans as well as the size and tractability of cats make them ideal for many experimental models. As such, a large database exists based on studies using cats as models of human disease.
As with dogs, the genetic diversity of the general cat population (and of some purpose-bred cats) has provided several valuable genetically based models of human disease. For example, a colony of hypertrophic cardiomyopathy Maine Coon cats is maintained at the University of California, Davis, and cats with mucopolysaccharidosis are maintained and studied at the University of Pennsylvania (Haskin 2009).
There are over 200 hereditary human diseases with correlates in cats (O’Brien et al. 2008). Following are a few illustrative examples.
Feline Immunodeficiency Virus
Cats are naturally susceptible to infection by feline immunodeficiency virus (FIV), which induces an immune suppressive disorder very similar to human immunodeficiency virus (HIV) infection in humans (Willett and Hosie 2008). A number of studies are investigating the mechanisms of FIV infection in cats as a model for HIV infection, and the cat models are also important in efforts to understand HIV in order to develop novel and more effective therapies for this devastating disease.
While it is possible to experimentally infect purpose-bred cats with FIV, the clinical manifestations of experimental infections differ from naturally occurring FIV infection (English et al. 1994). Indeed, even after 4 years of FIV infection specific pathogen-free (purpose-bred) cats do not exhibit chronic clinical disease (Torten et al. 1991). The differences between naturally occurring and induced FIV may be due to infectious cofactors in the random source animals (English et al. 1994; Willett and Hosie 2008).
FIV is the only naturally occurring model of acquired immunodeficiency syndrome (AIDS) (Dias et al. 2006). An additional advantage of FIV models in the study of AIDS is that this virus does not infect humans. For all these reasons, random source animals naturally infected with FIV represent a critical resource for understanding FIV, its sequelae, and its transmission between hosts.
Feline Interstitial Cystitis
Human interstitial cystitis, a serious bladder disorder characterized by pain, urinary frequency, and nocturnal urination (Roppolo et al. 2005), occurs at frequencies as high as 1 in 4.5 women (March et al. 2001). The causes for this disorder are not well understood. Domestic cats develop feline interstitial cystitis, which is clinically indistinguishable from the human disorder (Westropp and Buffington 2002). Random source cats are the only known spontaneously occurring animal models of the disease (Westropp and Buffington 2002).
Feline Infectious Peritonitis
This disorder, associated with vascular inflammation in a variety of organs, is almost always fatal. It is caused by an infectious agent in the coronavirus family, feline infectious peritonitis virus (FIPV) (Olsen 1993; Takano et al. 2008), which is thought to mutate from the more commonly found feline enteric coronavirus (FECV) (Vennema et al. 1998). FECV is very common in random source animals, but does not induce life-threatening disease (Olsen 1993). The conditions responsible for the mutation of FECV
to FIPV are not well understood, but appear to be associated with significantly increased viral replication in immunosuppressed animals (Haijema et al. 2004). Replication of FECV also radically increases when infected random source animals are placed in close association, such as in shelters (Pedersen et al. 2004).
FIPV pathogenesis is dependent on a mechanism known as antibody-dependent enhancement, in which host antibodies bind to the virus and the antibody-virus complex infects macrophages (Takano et al. 2008). Because antibody-dependent enhancement may be important in human viral diseases such as Dengue fever and HIV infection (Olsen 1993), FIPV-infected animals represent important resources in efforts to understand the pathogenesis of such diseases. Purpose-bred animals can be infected with FIPV and the virus can be cultured in feline kidney cells (Takano et al. 2008), but random source animals are a valuable initial resource for FIPV and its multiple strains (Olsen 1993), and represent models for understanding the process of mutation that produces a highly pathogenic virus from a related but far less virulent one.
IACUC AND PRINCIPAL INVESTIGATOR CONSIDERATIONS REGARDING THE USE OF RANDOM SOURCE ANIMALS FOR RESEARCH
The use in biomedical research of species that society regards as companion animals poses several unique challenges to principal investigators (PIs) and to the institutional animal care and use committees (IACUCs) that evaluate protocols describing research involving dogs and cats. While both groups have several forms of guidance available to them as they navigate the scientific and ethical issues inherent in experimental species selection and justification, the appropriate course to resolving these issues is not always clear.
As stated earlier, the Health Research Extension Act of 1985 mandated the establishment of guidelines for proper animal care by individuals and institutions that conduct research with funds provided by NIH or other federal sponsors. IACUCs are responsible for institutional oversight of animal care and treatment and, together with the PIs at institutions receiving NIH and other federal funds to support research, must comply with guidance found in the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Teaching (revised 2002). Although each of the nine Principles applies, several often receive special consideration when dogs and cats have been selected as research animals.
Principle III states, in part, “The animals selected for a procedure should be of an appropriate species and quality and the minimum number required to obtain valid results.” Research involving random source animals may
require more rigorous justification to satisfy the IACUC and the institutional community that these animal models are not only appropriate but also have scientific benefits that outweigh the use of purpose-bred animals. Thus, both PIs and IACUCs may need to consider strain, breed, and in some cases source when determining the appropriateness of animal models for particular studies.
The animal models may be defined as exploratory, explanatory, and/or predictive (Hau and Van Hoosier 2003); but cost alone has not routinely been a sufficient justification for the choice of an experimental model. The Guide (p. 12) and the AWR provide clear guidance related to cost and its inadequacy as the sole factor for determining the appropriateness of survival surgery models. Yet cost is a significant determinant; NIH grant budgets, for example, tend to favor the lower costs of random source animals over the higher costs of purpose-bred animals (see Chapter 4 for further discussion of the relative costs of animals from Class B dealers vs. purpose-bred animals).
As in all research involving animals, ethical and health concerns vary based on the condition of the animals when acquired and during their housing on the premises. Community concerns will vary based on the type of animal and its source. Although all animals used for research deserve humane treatment, additional training of IACUC members on the special challenges and opportunities associated with the use of random source animals may be warranted before the consideration and approval of any protocol involving animals of this type.
In particular, because random source animals have unknown health and care histories, potential health and animal welfare problems may be associated with their use, as discussed below. Not all IACUCs may have the collective experience to conduct a thorough risk-benefit analysis of the ramifications of using random source animals at their institution, so the use of random source animals requires teamwork, perhaps more so than in research involving purpose-bred animals whose health and care histories are known. PIs would be well advised to consult with institutional veterinarians, the IACUC, and the IACUC members who represent the concerns of the institutional community before, during, and after research involving random source animals.
IACUCs and PIs considering the use of random source animals may also face challenges related to Principle VII: “The living conditions of animals should be appropriate for their species and contribute to their health and comfort. Normally, the housing, feeding, and care of all animals used for biomedical purposes must be directed by a veterinarian or scientist trained and experienced in the proper care, handling, and use of the species being maintained or studied.” Institutions, and through them, veterinary staff and PI agree, in keeping with their PHS assurance, to uphold the high standards
of animal care and welfare. Thus, from the time of delivery of a research animal to the premises until the end of the research involving that animal, conditions of care and housing are subject to oversight by the IACUC and veterinarians periodically, and by the animal care and laboratory staff on a daily basis (9CFR 2.33(b)(3)). But institutions, IACUCs, and PIs are much less likely to oversee conditions at the sources from which they receive animals. There may be, either intentionally or unintentionally, different standards of care and animal housing at locations that provide animals compared with the research institutions that receive them. As with all other aspects of their animal programs, it would seem appropriate for institutions to periodically review their expectations about animal suppliers’ premises before obtaining animals from them.
Principle IX reminds PIs and IACUCs of their shared responsibilities in experimental model selection: “Where exceptions are required in relation to the provisions of these Principles, the decision should not rest with the investigators directly concerned but should be made, with due regard to Principle II, by an appropriate review group such as the IACUC. Such exceptions should not be made solely for the purposes of teaching or demonstration.” It is common practice for institutions to follow the U.S. Government Principles if PHS funds are received for research done on the premises. PHS Policy tends to be scrutinized more completely than the AWA. In addition, some institutions that accept PHS funding are more likely to be AAALAC accredited and therefore those institutions are subject to additional standards of oversight. Thus, if the use of animals of a given type or from a given source is considered (by the IACUC, the PI, or the institution itself) to differ from best veterinary and animal welfare practices, all of these groups should evaluate the possible justifications for exceptions to the U.S. Government Principles, PHS Policy, and perhaps the Guide.
In summary, IACUCs, PIs, and institutions face several challenges when studies are proposed and conducted that involve the use of random source companion animals, regardless of the sources of these animals. In such cases it is imperative that review committees evaluate the justification for animal use particularly carefully and thoroughly.
DELETERIOUS INFECTIOUS DISEASE ISSUES
Random source animals may be obtained from multiple sources, and the mingling of these animals may contribute to the spread of infectious disease. For example, 20 percent of dogs and 61 percent of cats acquired by Class B dealers come from shelters and pounds (USDA data submitted to the Committee, January 2009), and the health status of these animals is often unknown. Animals in shelters and pounds are more likely than purpose-bred animals to be exposed to outbreaks of infectious viral dis-
eases (e.g., canine distemper, canine parvovirus, canine parainfluenza virus, feline panleukopenia, feline calicivirus, and feline herpes virus). In addition, respiratory and intestinal diseases caused by viruses, bacteria, protozoa, and helminths are among the most common ailments that cause considerable morbidity and suffering for shelter animals.
The quality of care for shelter animals varies widely across the country. Shelters are not required to isolate, vaccinate, deworm, or provide treatment for illnesses in the animals (Miller and Zawistowski 2004) and, based on discussions with shelter experts (there is no published literature), the Committee found that many shelters do not have veterinarians on staff or even serving as advisors. Although some animals are very well cared for, they may be behaviorally abnormal, and they are almost certainly stressed. Furthermore, dogs and cats in shelters and pounds often have undocumented vaccination histories and frequently arrive at the shelter with compromised health—they may have heartworms, fleas, ticks, mites, lice, ringworm, or intestinal parasites, and/or a variety of disease agents that spread more readily than would normally be expected because the animals are mixed together. They may be placed into different types of group or communal housing, where unreliable sanitation practices contribute to disease spread. Research has shown that the longer animals stay in shelters and pounds, the more likely they are to develop respiratory disease (Edinboro et al. 2004). Even if vaccinated immediately upon entry, a stay of several days at a shelter puts animals at higher risk for respiratory disease because respiratory vaccines are not always effective in preventing infection. An additional consideration is that it is often not possible to detect animals that are incubating some infectious diseases because they appear clinically normal and diagnostic evaluation may be unavailable, incomplete, or misleading (as in instances of false negatives or positives).
To address these problems, the research institution or the Class B dealer (or both) conditions random source dogs and cats that enter research institutions (whether from shelters and pounds, Class B dealers, or other legal sources). The conditioning generally includes a period of quarantine, treatment for parasites, vaccination, deworming, and other health-related procedures that make the animal more suitable for research. Even so, the animals may still have health problems since not all infectious agents can be eliminated by antibiotics or deworming or prevented through vaccination. In contrast, purpose-bred animals are more likely to be microbiologically defined.
ZOONOTIC DISEASE HAZARDS AMONG RANDOM SOURCE ANIMALS
Some infectious disease agents associated with dogs and cats in the general pet population, and therefore among some random source animals,
pose a potential threat to humans. In the 2008 Compendium of Veterinary Standard Precautions for Zoonotic Disease Prevention in Veterinary Personnel (Appendix 1), the National Association of State Public Health Veterinarians lists 54 “zoonotic diseases of importance” in the United States; of these, 26 are associated with dogs and/or cats as the “most common species associated with transmission to humans” (2008). An earlier NRC report (1994, Table 2.1, p. 8) lists 27 “Selected Canine Zoonoses Causing Disease in Humans,” beginning with “acariasis” (mange) and ending with “yersiniosis” (see also NRC 1997, p. 95). And the World Health Organization Collaborating Center for New and Emerging Zoonoses lists numerous zoonotic agents, both common and rare, in domestic dogs and cats.9 Some common agents, such as Pasteurella spp., are present in the oral and nasal cavities of 12-92% of dogs and 52-99% of cats and are associated with infections from animal bites (Greene and Goldstein 2006). Other agents of concern include Bartonella henselae, the agent of “cat scratch disease” that is commonly carried by young cats; Salmonella and Campylobacter spp., which cause enteric disease; Sarcoptes spp., which causes scabies (in humans; called mange in animals); and Microsporidium (Microsporum) canis, which causes ringworm. Rabies represents a particularly serious zoonotic hazard among animals with unknown exposure and vaccination histories but is rare. Incidents of zoonoses in the research laboratory are fortunately rare, but recognition, control, and prevention of canine and feline zoonotic hazards are important aspects of institutional occupational safety programs (NRC 1997).
ADVERSE EFFECTS OF INFECTIOUS DISEASE ON RESEARCH
Exposure to infectious disease is a risk the research community can avoid. As discussed earlier, the use of random source animals for the study of naturally occurring infectious disease may be desirable, but in the other situations intercurrent infections may be deleterious to research. These considerations are generally taken into account by the individual investigator in concert with veterinary professionals at the research institution. Nonetheless, undetected (subclinical) infections can still compromise or confound research results. A recent study that documented canine exposure to three frequently reported tick-borne bacterial pathogens reported the results of molecular analysis and serology on 21 random source dogs from Class B dealers (Scorpio et al. 2008): the test results were positive in 17 dogs, but none showed any signs of clinical disease. The authors concluded that “Exposure to and potential for infection with these bacteria and other pathogens may contribute to blood and tissue alteration that
could confound experiments and lead to misinterpretation of data in canine models” (p. 23).
Heartworms (Dirofilaria immitus) are generally associated with dogs, but the incidence of infection in cats can be quite high in endemic areas—one study reported 76% prevalence in outdoor-housed cats in North Carolina (Atkins et al. 2005). Overt infection makes the animals unsuitable for most research, but even Dirofilaria-seropositive cats that lack adult worms in the heart and lung may have significant pulmonary disease, making them potentially unsuitable for cardiopulmonary studies (Browne et al. 2005).
ANIMAL WELFARE ISSUES
A basic understanding of the terms animal welfare, stress, and distress is essential to the discussion of humane issues and animal welfare in the context of this report and the Committee’s statement of task.
“Animal welfare” generally refers to the state of an animal and the extent to which it is faring well or ill in a particular situation or at a particular point in its life. Different experts give priority to different aspects of an animal’s state when assessing its welfare: some emphasize unpleasant or pleasant subjective feelings (Boissy et al. 2007; Dawkins 1980; Duncan 1993), while others focus on the animal’s ability to express “natural” or species-typical behavior (Rollin 1995) or its capacity to adapt to, or cope with, the demands of its environment (Broom and Fraser 2007). One thing all agree on is that there is no single, reliable measure of an animal’s welfare (Appleby 1999; Mason and Mendl 1993). Most animal welfare experts therefore advocate multiple measures of aspects that are likely to reflect an animal’s welfare (e.g., behavioral responses, physiological indicators, immune function) while at the same time recognizing that the final determination inevitably involves a degree of subjectivity (Dawkins 1980; Fraser 1995; Mason and Mendl 1993).
A recent NRC report (2008, p. 2) defines “stress” as a “real or perceived perturbation to an organism’s physiological homeostasis or psychological well-being.” Animals respond to such perturbations by displaying a “stress response,” characterized by behavioral and physiological efforts to restore homeostasis. Potential stressors may be physical or emotional and include overcrowding; changes in routine, diet, environment, temperature, or humidity; perceived threats to safety; sources of pain or discomfort; and malnutrition, illness, or physical restraint, among others.
A certain amount of stress is a normal part of any animal’s life and should not necessarily be considered detrimental to welfare. Stress should be regarded as a welfare problem only when the degree of perturbation is sufficiently acute or prolonged, and an animal’s capacity to restore homeostasis is exceeded. Many authorities now use the term “distress” to describe
the aversive negative state that arises when an animal is pushed to the limit of its ability to cope with, or adapt to, environmental stressors (NRC 2008), while the term “suffering” generally applies only to the conscious experience of highly aversive or unpleasant mental and emotional states, such as pain or fear (Dawkins 1998).
The question of whether random source dogs and cats experience a greater degree of stress and distress in the research laboratory setting than do purpose-bred animals cannot be answered directly as no published studies have addressed this question. Indirect evidence that the transition to life in laboratory housing may be stressful and distressing for former pets can, however, be derived from studies that have examined how pet dogs and cats respond to, and cope with, comparable transitions—for example, among pets relinquished to animal shelters, or those confined temporarily in boarding kennels, catteries, or veterinary hospital cages. Most such studies have found behavioral and physiological changes (e.g., elevated heart rate and glucocorticoid levels, reduced heart rate variability and white blood cell counts) consistent with the effects of moderate to severe stress. These responses may take 2 to 5 weeks to return to “normal” baseline levels, although some animals may remain in a distressed state for several months (Beerda et al. 1999a, b; Hennessy et al. 2001; Kessler and Turner 1997, 1999; McCobb et al. 2005; Rochlitz et al. 1998; Siracusa et al. 2008; Stephen and Ledger 2006; Väisänen et al. 2005).
Chronic stress is immunosuppressive and reduces both cell-mediated and humoral immunity, thus increasing susceptibility to infectious disease, vasodepressive syncope, blood clots, coronary vasoconstriction, and other effects (Gregory 2004). A variety of factors may contribute to these outcomes, including the stressful effects of physical confinement and lack of stimulation, loss of social companions, exposure to unfamiliar people or conspecifics, and lack of control over environmental stressors (Beerda et al. 1999a, b; Carlstead et al. 1993; Hubrecht 1995; McCrave 1991). Because some random source dogs and cats are former pets or strays and therefore not used to prolonged cage confinement, it is reasonable to infer that they may have more difficulty adjusting to laboratory conditions than purpose-bred animals (see British Veterinary Association Animal Welfare Foundation et al. 2004).
In summary, based on the limited available evidence, random source dogs and cats used for research probably endure greater degrees of stress and distress compared to purpose-bred animals. This conclusion has implications both for the welfare of random source animals and for their reliability as research models. Stress and distress are known to significantly alter animals’ physiological and behavioral responses to experimental manipulations, and will therefore affect the quality of the scientific results obtained from such animals (NRC 2008; Reinhardt 2004).
American Humane Shoptalk. 1988. April/May. 6(3).
An, Y. and Friedman, Y., eds. 1999. Animal Models in Orthopedic Research. CRC Press: Boca Raton, FL. pp. 505-526.
Antzelevitch, C. December 3 2008. Letter submitted to the Committee. Masonic Medical Research Laboratories.
Anyukhovsky, E. P., E. A. Sosunov, P. Chandra, T. S. Rosen, P. A. Boyden, P. Danilo, and M. R. Rosen. 2005. Age-associated changes in electrophysiologic remodeling: a potential contributor to initiation of atrial fibrillation. Cardiovascular Research. 66:353-363.
Appleby, M. C. 1999. What should we do about animal welfare? Oxford: Blackwell Scientific.
Arnoczky, S. P., G. B. Tarvin, and J. L. Marshall. 1982. Anterior cruciate ligament replacement using patellar tendon. An evaluation of graft revascularization in the dog. Journal of Bone Joint Surgery of America. 64:217-224.
Atkins, C., A. Moresco, and A. Litster. 2005. Prevalence of naturally occurring Dirofilaria immitus infection among non-domestic cats housed in an area in which heartworms are endemic. Journal of the American Veterinary Medical Association. 227:139-143.
Basso, C., P. R. Fox, K. M. Meurs, J. A. Towbin, A. W. Spier, F. Calabrese, B. J. Maron, and G. Thiene. 2004. Arrhythmogenic right ventricular cardiomyopathy causing sudden cardiac death in Boxer dogs. Circulation. 109:1180-1185.
Beerda, B., M. B. Schilder, J. Van Hooff, H. W. de Vries, and J. A. Mol. 1999a. Chronic stress in dogs subjected to social and spatial restriction I. Behavioral response. Physiology and Behavior. 66:233-242.
Beerda, B, M. B. Schilder, J. Van Hooff, H. W. de Vries, and J. A. Mol. 1999b. Chronic stress in dogs subjected to social and spatial restriction. 2. Hormonal and immunological responses. Physiology and Behavior. 66:243-254.
Blumenstock, D. A., F. D. Cannon, V. L. Vlahovic, and H. D. Alpern. 1981. Transplantation of the lung from mongrel dogs into DLA-nonidentical beagles. Transplant Proceedings. Mar 13 (1 Pt 2):863-9.
Boissy, A., G. Manteuffel, M. B. Jensen, R. O. Moe, B. Spruijt, L. J. Keeling, C. Winkler, B. Forkman, I. Dimitrov, J. Langbein, M. Bakken, I. Veissier, and A. Aubert. 2007. Assessment of positive emotions in animals to improve their welfare. Physiology and Behavior 92:375-397.
British Veterinary Association Animal Welfare Foundation/FRAME/RSPCA/UFAW Joint Working Group on Refinement. 2004. Refining Dog Husbandry and Care. Laboratory Animals. 38 (Suppl. 1):1-94.
Broom, D. M. and A. F. Fraser. 2007. Domestic Animal Behaviour and Welfare, 4th Edition. Wallingford, Oxford: CABI.
Browne, L. E., T. D. Carter, J. K. Levy, P. S. Snyder, and C. M. Johnson. 2005. Pulmonary arterial disease in cats seropositive for Dirofilaria immitus but lacking adult heartworms in the heart and lungs. American Journal of Veterinary Research. 66:1544-1549.
Canine Inherited Disorders Database. 2004. http://www.upei.ca/~cidd/intro.htm
Carlstead, K., J. L. Brown, and W. Strawn. 1993. Behavioral and physiological correlates of stress in laboratory cats. Applied Animal Behaviour Science. 38:143-158.
Cook, S. D., J. E. Dalton, E. H. Tan, T. S. Whitecloud, and D. C. Rueger. 1994. In vivo evaluation of recombinant human osteogenic protein (rhOP-1) implants as a bone graft substitute for spinal fusion. Spine. 19(15):1655-1663.
CVMBS (Colorado State University College of Veterinary Medicine and Biomedical Sciences). 2006a. Shelter Derived Animal Use Guidance Statements Prohibition Against Use of Shelter-Derived Animals for Research or Teaching. Revised March 2006. http://www.cvmbs.colostate.edu/cvmbs/ShelterAnimalUse.htm
CVMBS. 2006b. Shelter Derived Animal Use for Research and Teaching Prohibition Against Use of Shelter-Derived Animals for Research or Teaching Guidance Statements. Revised March 2006. http://www.cvmbs.colostate.edu/cvmbs/ShelterAnimalGuidance.htm
Dawkins, M. S.1980. Animal Suffering: The Science of Animal Welfare. London: Chapman Hall.
Dawkins, M. S.1998. Evolution and animal welfare. Quarterly Review of Biology. 73:305-328.
Dias, A. S., M. J. Bester, R. F. Britz, and Z. Apostolides. 2006. Animal models used for the evaluation of antiretroviral therapies. Current HIV Research. 4:431-446.
Dun, W., T. Yagi, M. R. Rosen, and P. A. Boyden. 2003. Calcium and potassium currents in cells from adult and aged canine right atria. Cardiovascular Research. 58:526-534,
Duncan, I. J. H.1993. Welfare is to do with what animals feel. Journal of Agricultural and Environmental Ethics. 6 (Supp. 2):8-14.
Edinboro, C. H., M. P. Ward, and L. T. Glickman. 2004. A placebo-controlled trial of two intranasal vaccines to prevent tracheobronchitis (kennel cough) in dogs entering a humane shelter. Prevent Veterinary Medicine. 62(2):89-99.
English, R. V., P. Nelson, C. M. Johnson, M. Nasisse, W A. Tompkins, and M. B. Tompkins. 1994. Development of clinical disease in cats experimentally infected with feline immunodeficiency virus. Journal of Infectious Disease. 170:543-552.
Ferrari, A. U., A. Radaelli, and M. Centola. 2003. Aging and the cardiovascular system. Journal of Applied Physiology. 95:2591-2597.
Fraser, D.1995. Science, values and animal welfare: Exploring the “inextricable connection”. Animal Welfare. 4:103-117.
Frick, S. L., E. N. Hanley, R. A. Meyer, W. K. Ramp, and T. M. Chapman. 1994. Lumbar intervertebral disc transfer. A canine study. Spine. 19:1826-1834.
Fuller, G. A., B. Sabahattin, R. L. Hamlin, M. Yamaguchi, and P. J. Reiser. 2007. Increased myosin heavy chain-b with atrial expression of ventricular light chain-2 in canine cardiomyopathy. Journal of Cardiac Failure. 13(8):680-686.
Goralska, M., L. N. Fleisher, and M. C. McGahan. 2007. Ferritin H- and L-chains in fiber cell canine and human lenses of different ages. Investigative Ophthalmology and Visual Science. 48:3968-3975.
Goralska, M., S. Nagar. C. M. Colitz, L. N. Fleisher, and M. C. McGahan. 2009. Changes in ferritin H- and L-chains in canine lenses with age-related nuclear cataract. Investigative Ophthalmology and Visual Science. 50:305-310.
Greene, C. and E. C. Goldstein. 2006. Bite wound infections. In: Infectious Diseases of the Dog and Cat. C. Greene, ed. Saunders Elsevier. p. 499.
Gregory, N. G. 2004. Physiology and behavior of animal suffering. Oxford, UK: Blackwell Science. 268 pp.
Greis, P. 2001. The influence of tendon length and fit on the strength of a tendon-bone tunnel. A complex biomechanical and histologic study in dogs. American Journal of Sports Medicine. 29:493-497.
Haijema, B. J., H. Volders, and P. J. Rottier. 2004. Live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis. Journal of Virology. 78:3863-3871.
The Harris Poll®. 2007. Pets Are “Members of the Family” and Two-Thirds of Pet Owners Buy Their Pets Holiday Presents #120, December 4, 2007. http://www.harrisinteractive.com/harris_poll/index.asp?PID=840
Hartman, H. A. 1989. Spontaneous extramural coronary arthritis in dogs. Toxicologic Pathology. 17(1 Pt 2):138-144.
Hasegawa, K., C. H. Turner, C. Jie, and D. B. Burr. 1995. Effect of disc lesion on microdamage accumulation in lumbar vertebrae under cyclic compression loading. Hip Society Meeting 1994. Clinical Orthopaedics and Related Research. 311:190-198.
Haskin, M. 2009. Gene therapy for lysosomal storage diseases (LSDs) in large animal models. ILAR Journal. 50(2):112-121.
Hau, J. and G. L. Van Hoosier eds. 2003. Handbook of Laboratory Animal Science, 2nd Ed. Vol. 2. Boca Raton, FL: CRC Press, p. 2.
Hennessy, M. B., V. L. Voith, S. J. Mazzei, J. Buttram, D. D. Miller, and F. Linden. 2001. Behavior and cortisol levels of dogs in a public animal shelter, and an exploration of the ability of these measures to predict problem behavior after adoption. Applied Animal Behaviour Science. 73:217-233.
Hubrecht, R. C. 1995. The welfare of dogs in human care. In: The Domestic Dog: Its Evolution, Behavior and Interactions with People. J. A. Serpell, ed. Cambridge: Cambridge University Press. pp. 179-198.
Hunter, C. J., J. R. Matyas, and N. A. Duncan. 2004. Cytomorphology of notochordal and chondrocytic cells from the nucleus pulposus: a species comparison. Journal of Anatomy. 205(5):357–362.
Kaczka, D. W., D. N. Hager, M. L. Hawley, and B. A. Simon. 2005. Quantifying mechanical heterogenicity in canine acute lung injury. Anesthesiology. 103(2):306-312.
Kass, P. H., J. C. New, J. M. Scarlett, and M. O. Salman. 2001. Understanding animal companion surplus in the United States: Reliquishment of nonadoptables to animal shelters for euthanasia. Journal of Applied Animal Welfare Science. 4(4):237-248.
Katsuura, A. and S. Hukuda. 1994. Experimental study of intervertebral disc allografting in the dog. Spine. 19(21):2426-2432.
Kessler, M. R. and D. C. Turner. 1997. Stress and adaptation of cats (Felis silvestris catus) housed singly, in pairs, and in groups in boarding catteries. Animal Welfare. 6:243-254.
Kessler, M. R. and D. C. Turner. 1999. Socialization and stress in cats (Felis silvestris catus) housed singly and in groups in animal shelters. Animal Welfare. 8:15-26.
Lee, K. W., T. H. Everett, D. Rahmutula, J. M. Guerra, E. Wilson, C. Ding, and J. E. Olgin. 2006. Pirfenidone prevents the development of a vulnerable substrate for atrial fibrillation in a canine model of heart failure. Circulation. 114(16):1703-1716.
Lupo, M. and R. Storb. 2007. Five decades of progress in haematopoietic cell transplantation based on preclinical canine model [Review]. Veterinary and Comparative Oncology. 5(1):14-30.
MacEwen, E. G. 1990. Spontaneous tumors in dogs and cats: models for the study of cancer biology and treatment. Cancer and Metastasis Reviews. 9(2):125-36.
March, P., B. Teng, J. Westropp, and T. Buffington. 2001. Effects of resiniferatoxin on the neurogenic component of feline interstitial cystitis. Urology. 57:114.
Mason, G. J. and M. Mendl. 1993. Why is there no simple way of measuring animal welfare? Animal Welfare. 2: 301-320.
McCobb, E. C., G. Patronek, A. Marder, J. D. Dinnage, and M. S. Stone. 2005. Assessment of stress levels among cats in four animal shelters. Journal of the American Veterinary Medical Association. 226:548-555.
McCrave, A. E. 1991. Diagnostic criteria for separation anxiety in the dog. Veterinary Clinics of North America: Small Animal Practice. 21:247-255.
Miller, L., and S. Zawistowski, eds. 2004. Shelter Medicine for Veterinarians and Staff. Ames, Iowa: Blackwell Publishing.
National Association of State Public Health Veterinarians. 2008. The Compendium of Veterinary Standard Precautions for Zoonotic Disease Prevention in Veterinary Personnel. Appendix 1.
Neumann, J. and T. Bilzer. 2005. Evidence for MHC I-restricted CD8+ T-cell-mediated immunopathology in canine masticatory muscle myositis and polymyositis. Muscle and Nerve. 33(2):215-224.
Nguyen-minh, C., L. Riley, K.-C. Ho, R. Xu, H. An, and V. M. Haughton. 1997. Effect of degeneration of the intervertebral disk on the process of diffusion. American Journal of Neuroradiology. 18:435-442.
Nichols, T. C., A. M. Dillow, H. W. G. Franck, E. P. Merricks, R. A. Raymer, D. A. Bellinger, V. R. Arruda, and K. A. High. 2009. Protein replacement therapy and gene transfer in canine models of hemophilia A, hemophilia B, von Willebrand disease, and factor VII deficiency. ILAR Journal. 50(2):144-167.
NRC (National Research Council). 1994. Dogs – Laboratory Animal Management. Washington: National Academy Press. p 8.
NRC. 1997. Occupational Health and Safety in the Care and Use of Research Animals. Washington: National Academy Press.
NRC. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. p. 10.
NRC. 2008. Recognition and Alleviation of Distress in Laboratory Animals. Washington, DC: The National Academies Press.
O’Brien, S. J., W. Johnson, C. Driscoll, J. Pontius, J. Pecon-Slattery, and M. Menottti-Raymond. 2008. State of cat genomics. Trends Genetics. 24:268-279.
Olsen, C. W. 1993. A review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination. Veterinary Microbiology. 36:1-37.
Opii, W. O., G. Joshi, E. Head, N. W. Milgram, B. A. Muggenburg, J. B. Klein, W. M. Pierce, C. W. Cotman, and D. A. Butterfield. 2008. Proteomic identification of brain proteins in the canine model of human aging following a long-term treatment with antioxidants and a program of behavioral enrichment: relevance to Alzheimer’s disease. Neurobiology of Aging. 29:51-70.
Ostrander, E. A. and R. K. Wayne. 2005. The canine genome [Review]. Genome Research. 15:1706-1716.
Parsons, R. E., M. L. Marin, F. J. Veith, L. A. Sanchez, R. T. Lyon, W. D. Suggs, P. L. Faries, and M. L. Schwartz. 1996. Fluoroscopically assisted thromboembolectomy: an improved method for treating acute arterial occlusions. Annals of Vascular Surgery. 10(3):201-210.
Pedersen, N. C., R. Sato, J. E. Foley, and A. M. Poland. 2004. Common virus infections in cats, before and after being placed in shelters, with emphasis on feline enteric coronavirus. Journal of Feline Medicine and Surgery. 6:83-88.
Reinhardt, V. 2004. Common husbandry-related variables in biomedical research with animals. Laboratory Animals. 38:213-235.
Reising, C. A., A. Chendrasekhar, P. L. Wall, N. F. Paradise, G. A. Timberlake, Rochlitz, I., A. L. Podberscek, and D. M. Broom. 1998. Welfare of cats in a quarantine cattery. Veterinary Record. 143:35-39.
Rochlitz, I., A. L. Podberscek, and D. M. Broom. 1998. Welfare of cats in a quarantine cattery. Veterinary Record. 143: 35-39.
Rollin, B. E. 1995. Farm Animal Welfare: Social, Bioethical and Research Issues. Ames: Iowa State University Press.
Roppolo, J. R., C. Tai, A. M. Booth, C. A. Buffington, W. C. de Groa, and L. A. Birder. 2005. Bladder Adelta afferent nerve activity in normal cats and cats with feline interstitial cystitis. Journal of Urology. 173:1011-1015.
Russell, W. M. S. and R. L. Burch. 1959. The Principles of Humane Experimental Technique. London: Methuen & Co. Reprinted by Universities Federation for Animal Welfare, UK. 1992.
Sasajima, T., V. Bhattacharya, M. H. Wu, Q. Shi, N. Hayashida, and L. R. Sauvage. 1999. Morphology and histology of human and canine internal thoracic arteries. Annals of Thoracic Surgery. 68(1):143-148.
Scorpio, D. G., L. M. Wachtman, R. S. Tunin, N. C. Barat, J. W. Garyu, and J. S. Dumler. 2008. Retrospective clinical and molecular analysis of conditioned laboratory dogs (Canis familiaris) with serologic reactions to Ehrlichia canis, Borrelia burgdorferi, and Rickettsia rickettsii. Journal of the American Association for Laboratory Animal Science. 47(5):23-28.
Serpell, J. A. and J. A. Jagoe.1995. Early experience and the development of behaviour. In: The Domestic Dog: Its Evolution, Behaviour and Interactions with People, J. A. Serpell ed. pp. 80-102. Cambridge: Cambridge University Press.
Siracusa, C., X. Manteca, J. Cerón, S. Martínez-Subiela, R. Cuenca, S. Lavín, F. Garcia, and J. Pastor. 2008. Perioperative stress response in dogs undergoing elective surgery: Variations in behavioural, neuroendocrine, immune and acute phase response. Animal Welfare. 17:259-273.
Siwak-Tapp, C. T., E. Head, B. A. Muggenburg, N. W. Milgram, and C. W. Cotman. 2007. Neurogenesis decreases with age in the canine hippocampus and correlates with cognitive function. Neurobiology of Learning and Memory. 88:249-259.
Siwak-Tapp, C. T., E. Head, B. A. Muggenburg, N. W. Milgram, and C. W. Cotman. 2008. Region-specific neuron loss in the aged canine hippocampus is reduced by enrichment. Neurobiology of Aging. 29:39-50.
Sleeper, M. M., L. T. Bish, and H. L. Sweeney. 2009. Gene therapy in large animal models of human cardiovascular genetic disease. ILAR Journal. 50(2):199-205.
Smith, G. N., S. L. Myers, K. D. Brandt, and E. A. Mickler. 1998. Effect of intraarticular hyaluronan injection in experimental canine osteoarthritis. Arthritis and Rheumatism. 41(6):976-985.
Smucker, M. L., S. Kaul, J. A. Woodfield, J. C. Keith, S. A. Manning, and J. A. Gascho. 1990. Naturally occurring cardiomyopathy in the Doberman pinscher: a possible large animal model of human cardiomyopathy? Journal of the American College of Cardiology. 16(1):200-206.
Stephen, J. M. and R. A. Ledger. 2006. A longitudinal evaluation of urinary cortisol in kenneled dogs, Canis familiaris. Physiology and Behavior. 87:911-916.
Stieger, K., E. Lhériteau, P. Moulier, and F. Rolling. 2009. AAV-mediated gene therapy for retinal disorders in large animal models. ILAR Journal. 50(2):206-224.
Studzinski, C. M., L. A. Christie, J. A. Araujo, W. M. Burnham, E. Head, C. W. Cotman, and N. W. Milgram. 2006. Visuospatial function in the beagle dog: an early marker of cognitive decline in a model of human aging and dementia. Neurobiology of Learning and Memory. 86:197-204.
Swindle, M. M. and R. J. Adams, eds. 1988. Experimental Surgery and Physiology: Induced Animal Models of Human Disease. Hagerstown, MD: Williams & Wilkins.
Takano, T., C. Kawakami, S. Yamada, R. Satoh, and T. Hohdatsu. 2008. Antibody-dependent enhancement occurs upon re-infection with the identical serotype virus in feline infectious peritonitis virus infection. The Journal of Veterinary Medical Science/The Japanese Society of Veterinary Science. 70:1315-1321.
Taylor, D. G., L. D. Parilak, M. M. LeWinter, and H. J. Knot. 2004. Quantification of the rat left ventricle force and Ca2+ -frequency relationships: similarities to dog and human. Cardiovascular Research. 61:77-86.
Thomas, E. D. 1990. Bone marrow transplantation – past, present and future. Nobel lecture, December 8 1990. In: Les Prix Nobel: The Nobel Prizes 1990. T. Frangsmyr, ed. Stockholm, Sweden: Nobel Foundation. pp. 581-582.
Torten, M., M. Franchini, J. E. Barlough, J. W. George, E. Mozes, H. Lutz, and P. C. Pedersen. 1991. Progressive immune dysfunction in cats experimentally infected with feline immunodeficiency virus. Journal of Virology. 65:2225-2230.
Tune, J. D., K. N. Richmond, M. W. Gorman, R. A. Olsson, and E. O. Feigl. 2000. Adenosine is not responsible for local metabolic control of coronary blood flow in dogs during exercise. American Journal of Physiology Heart Circulation Physiology. 278:H74-H84.
Turner, D. C.2000. The human-cat relationship. In: The Domestic Cat: The Biology of Its Behaviour, 2nd edition, D. C. Turner and P. P. G. Bateson, eds. pp. 194-206. Cambridge: Cambridge University Press.
Väisänen, M. A., A. E. Valros, E. Hakaoja, M. R. Raekallio, and O. M. Vainio. 2005. Preoperative stress in dogs: a preliminary investigation of behaviour and heart rate variability in healthy hospitalized dogs. Veterinary Anaesthesia and Analgesia. 32:158-167.
Vennema, H., A. Poland, J. Foley, and N. C. Pedersen. 1998. Feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses. Virology 243:150-157.
Wang, Z., J. S. Chamberlain, S. J. Tapscott, and R. Storb. 2009. Gene therapy in large animal models of muscular dystrophy. ILAR Journal. 50(2):187-198.
Westropp, J. L., and C. A. Buffington. 2002. In vivo models of interstitial cystitis. Journal of Urology. 167:694-702.
The White House. 2006. The Federal Response to Hurricane Katrina Lessons Learned. February 2006. Appendix A. http://220.127.116.11/docview.asp?docid=6457&locid=97
Willett, B. J., and M.J. Hosie. 2008. Chemokine receptors and co-stimulatory molecules: unravelling feline immunodeficiency virus infection. Veterinary Immunology and Immunopathology. 123:56-64.
Williams, R. E., D. A. Kass, Y. Kawagoe, P. Pak, R. S. Tunin, R. Shah, A. Hwang, and A. M. Feldman. 1994. Endomyocardial gene expression during development of pacing tachycardia-induced heart failure in the dog. Circulation Research. 75:615-623.
Wolfe, J. H. 2009. Gene therapy in large animal models of human genetic diseases. ILAR Journal 50(2):107-242.
Zawistowski, S. 2008. Companion Animals in Society. USA.: Thomson Delmar Learning. p. 81.