ILAR Journal Online, Volume 39(4) 1998: Opportunistic Infections in Laboratory Rats and Mice

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ILAR Journal V39(4) 1998
Opportunistic Infections in Laboratory Rats and Mice

Reflections on Future Needs in Research with Animals
Diane J. Gaertner, Lela K. Riley, and Dale G. Martin

Diane S. Gaertner, D.V.M., is Director of the Institute for Animal Studies and Associate Professor of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York. Lela K. Riley, Ph.D., is a Molecular Biologist in the Department of Veterinary Pathobiology and the Research Animal Diagnostic & Investigative Laboratory at the College of Veterinary Medicine, University of Missouri at Columbia, Columbia, Missouri. Dale G. Martin, D.V.M., Ph.D., is a Colonel in the Veterinary Corps and Director of the Division of Veterinary Medicine, Walter Reed Army Institute of Research, Washington, DC.

The advent of new scientific tools such as genetically manipulated rodents and mapping of the mouse genome has resulted in the use of reduced numbers of larger animals and in explosively growing populations of rodents (especially mice). At the same time, factors such as public concerns for animal wellness, availability, and costs for many traditionally used larger species have made their continued use more difficult to justify. As the total number of mice and rats used for research has increased, many individual rodents have become more valuable and many rodents are now more susceptible to disease. Because genetically manipulated rodents are enabling scientific investigators to answer a whole new universe of questions in vivo, we expect that the growth of rodent usage will continue for the next 5 to 10 yr. The purpose of this article is to describe the animal-related resources that we will need as we enter the 21st century to continue the progress of biomedical research. A discussion of future animal care needs is appropriate in this ILAR Journal issue devoted to opportunistic infections because many genetically manipulated rodents---the tools of the future---will be exquisitely sensitive to opportunistic infections. We postulate that major needs will exist for animals, staffing, and infrastructure to facilitate scientific progress between the time of this writing and 2010.

ANIMAL-BASED RESEARCH TRENDS

Visible trends that characterize the use of research animals in the 21st century include the evolving use of genetically manipulated mice and other individual research animals, the continued need for investigators to breed their own rodents in-house, the emergence of zebrafish as a key vertebrate model, and the increasingly sophisticated role of the veterinarian as a member of the team of researchers who utilize experimental animals. We briefly discuss each of these trends below.

Genetically Manipulated Animals

The dominant trend in animal-based research will involve genetically manipulated animals (especially mice) in an attempt to answer basic questions of mammalian biology and to model human diseases (Sharp and Davisson 1994). Transgenic knock-out and knock-in techniques now permit scientists to finally begin to answer crucial basic questions in an in vivo mammalian model. Because this technology is still very new at the time of this writing, scientists have so far asked only a small proportion of the questions these animals can help to answer. Thus we are moving rapidly toward a sustained increase in the numbers of mice that will be used for research.

Concurrently, many individual rodents have become more valuable because of their unique genetic manipulations and the corresponding miniaturization of research techniques. Among genetically manipulated mice, founder and early generation individuals are frequently irreplaceable. These animals are also frequently more susceptible to disease due to unexpected effects of genetic manipulation, such as partial or complete immunodeficiency secondary to insertion of transgenic constructs in genes producing or regulating immunoactive proteins. Thus at the time of this writing, genetically manipulated rodents require high-quality housing conditions and intensive health monitoring beyond the requirements of less
susceptible rodents (Cork and others 1997).

In-house Animal Breeding

Although healthy, inexpensive, pathogen-free rodents are readily available for purchase, we expect that many investigators will need to breed their animals in-house, rather than purchasing them from commercial vendors. This is a secondary effect of producing unique genetically manipulated founder animals. After producing founder animals, investigators must grow a colony of progenitors to study gene effects. Frequently, different mutations must be combined via multiple generations of breeding to examine biologically relevant questions within complex biochemical pathways, such as carcinogenesis. The recently increased need for more animals and multiple generations of in-house breeding to examine multigene effects leads us to expect that this increase will continue in the foreseeable future. Development and maintenance of many breeding colonies has slowed efforts aimed at eradication of common murine infectious agents. Although much progress has been made in eradication of common laboratory animal diseases due to agents such as Mycoplasma pulmonis and Sendai virus, other infectious agents such as murine coronaviruses (mouse hepatitis virus and rat coronaviruses) and pinworms remain highly prevalent (Jacoby and Lindsey 1998) and continue to threaten animal health.

An important new trend is the emergence of zebrafish as a key vertebrate model. Scientists are using the zebrafish system, with its genetic versatility and sophisticated embryology, to enhance our understanding of developmental biology, neurobiology, teratology, and toxicology (NRC 1998). The zebrafish system is useful for genetic experiments that cannot be accomplished with other vertebrate models and for offering a valuable complement to investigators utilizing other vertebrate model systems. We expect the use of zebrafish to expand as more uses for this versatile system are devised. To fully utilize zebrafish, the training of laboratory animal scientists, comparative pathologists, and research scientists should be expanded to include the uses, care, and diseases of this rapidly emerging experimental animal.

TRAINING AND EDUCATION

Veterinarian's Role

Although the role of the veterinarian as part of the research team is evolving and becoming more sophisticated, the central focus continues to be maintenance of healthy animals in a constant environment to permit investigators to generate valid animal-based research findings. Investigators must decipher the changes caused by their manipulations from abnormalities due to environment or disease. These growing mouse populations, the increased value and susceptibility of individual mice, the expanding exchange of animals between institutions, and the persistence of significant infectious agents makes this an ever more challenging job. We are concerned that the growing regulatory role of many veterinarians will slow the growth of knowledge about research animal diseases, environmental factors, and how such factors reduce the validity of research results. It is essential for laboratory animal veterinarians to ensure that the confounding effects of nongenetic factors are distinguished from genetic effects. Teams of investigators and veterinarians utilizing animals also need to improve diagnostic techniques supporting continued improvements in animal health and to assist in phenotyping of genetically altered experimental animals.

Additional trained people who will be key to optimal care and utilization of the experimental animals of the 21st century are described below and include skilled veterinary specialists, trained animal caretakers, knowledgeable scientists, and an educated public.

Veterinary Specialists

Specialists in veterinary medicine include laboratory animal veterinarians, comparative pathologists, veterinary pathologists, and others who have the training and knowledge to contribute to scientific progress through service activities and scientific research. Because the experimental animal population has changed, veterinarians providing oversight and health care for these animals must be well trained in the specialty of laboratory animal medicine. Although the total number of mice being used has grown explosively, the number of other animals used in research is decreasing (Crawford 1996). Experimental animal populations at major biomedical institutions increasingly consist of rodents. The numbers of other animal species being used, such as dogs, cats, swine, rabbits, and small rodents other than rats and mice, are stable or are shrinking. Larger species are used when unique species characteristics or body size preclude the use of mice or rats. Larger species will continue to be required for certain types of studies, such as development of surgical models, testing of medical devices, and investigations of fetal physiology and cerebral blood flow. In addition, regulatory requirements dictate that nonrodent species are needed in certain studies, such as toxicology and pharmacokinetics studies in which dogs or nonhuman primates are used. The use of nonhuman primates will remain essential for the biomedical community. Further consolidating resources will likely result in a decreased number of institutions using nonhuman primates; however, the actual numbers of nonhuman primates may increase. Nonhuman primates continue to be used because of their unique similarity to humans in, for example, disease susceptibility (AIDS, malaria, and hepatitis research) and neuroanatomy (Johnson 1995).

Instruction about pet and farm animal health care, the central emphasis of veterinary curricula, does not prepare veterinarians to deal with the specialized health problems and colony health management that are the major health care needs for large rodent and nonhuman primate colonies. Typical veterinary education also fails to train students in the anesthetic and critical care procedures necessary for dealing with larger domestic species in a research setting. Training for these activities must be available and must emphasize health care and health maintenance for rodents and nonhuman primates, with specialized knowledge of rodent and primate diseases, noninfectious conditions, and practical epidemiology under the conditions found in colonies of experimental animals. Although such specialized information can be acquired by extensive "hands-on" experience, several years of supervised training in laboratory animal and comparative medicine provide optimal training for veterinarians to enter a research environment. Although a recent survey predicted that supply and demand for laboratory animal veterinarians were at a steady state (Weigler and others 1997), we are concerned that opportunities for high-quality training have become fewer as federal support has shifted from combined research and clinical training to exclusively research training for veterinarians (Cork and others 1997). The absence of programs that combine high-quality clinical training and research training may limit the number of veterinarians able to provide optimal veterinary care and oversight for tomorrow's uniquely valuable, expanding, immunocompromised animal populations. This lack of specifically trained veterinarians may endanger the health of laboratory animals and ultimately jeopardize the progress of biomedical research.

While rodent populations bred in-house have been growing (as described in Trends), there has been an exponential growth of interinstitutional and international exchange of genetically modified rodents. Although European organizations have proposed quality assurance standards, these standards have not been adopted uniformly, and no such standards are used within the United States. For this reason, veterinary expertise is essential to provide the quarantine and rederivation services that protect in-house rodent populations from infectious agents. Rodent populations must be protected because even subclinical infection has been shown to unexpectedly alter research results (Bhatt and others 1986; NRC 1991). Quarantine and rederivation services, which require veterinarians with advanced training and experience as well as specialized animal facilities and operating procedures, are among the services most crucially needed by research institutions at the time of this writing.

Trained veterinarians will be needed with skills in investigative phenotyping of new genetically manipulated animals (NRC 1998). Veterinary clinicians and, to an even greater extent, veterinary pathologists will be integral to the development and assessment of the phenotypic effects of transgene insertion and gene knock-out. Ideally, veterinary pathologists with comprehensive knowledge of disease processes, human diseases, and rodent diseases will consult with scientists as they develop models to decode genetic effects and model aspects of human diseases. There exist relatively few veterinary/comparative pathologists with training and experience in these areas, and the expanding market for these skills is expected to far exceed the supply of trained individuals as increasing numbers of rodent models must be characterized. Creation of centers that can perform comprehensive phenotyping of genetically manipulated animals will be expensive yet extremely valuable for scientific progress. We expect that the resources of a single institution will not usually be adequate to meet the full range of potential phenotyping needs; thus a cooperative network utilizing today's methods of virtual communication may better serve these needs.

Scientists

Trained scientists are essential to gain maximal benefit from the numerous new rodent models to be created in the next decade (NRC 1998). Many scientists who previously used only in vitro methods are now using animals. These individuals and their collaborators require training even in the most basic of whole animal experimental techniques and animal handling (Cork and others 1997). Scientists must understand that phenotypic effects of genetic alterations must be distinguished from "background" changes, such as those due to mouse-strain effects, environmental factors, and intercurrent infections/infestations. In an ideal world, rodents would be free of these changes. However, as detailed in the article by Jacoby and Lindsey (1998), viral, bacterial, and parasitic agents have not been eliminated and continue to affect many experimental colonies.

Knowledgeable scientists will advocate that animals should be free of infectious agents that may confound research results. Scientists will understand the scientific value of state-of-the-art rodent quality assurance testing and of veterinary input into phenotype investigation and will support these costs through per diem, fee-for-service, and collaborative mechanisms. Knowledgeable investigators will demand diagnostic testing of laboratory animals for infectious agents and will expect their animals to be maintained disease free. Reputable journals will insist that information regarding the pathogen status of the experimental animals be included routinely as part of the Methods section. Investigators and staff also need ongoing education in handling, behavior, and breeding of rodents as they strive to produce new rodent models. Training regarding techniques for back-crossing, intercrossing, and genetic monitoring must be made available to investigators and technicians unitizing complex breeding methods to study multigene interactions.

Animal Caretakers

In addition to veterinarians and veterinary pathologists, animal caretakers are important members of the optimal animal care team for genetically manipulated animals, which will be crucial to scientific progress between now and 2010. Animal caretakers provide a vital link among laboratory animals, scientists, and veterinarians.

Trained animal caretakers, such as those certified by the American Association for Laboratory Animal Science, have observation skills that improve animal care by early detection of clinical problems due to genetic factors or to infectious agents. As rodent populations continue to grow, the animal caretaker will be the primary individual to assess daily animal wellness and behavior as each animal is viewed during daily checks and routine husbandry tasks. Additionally, proper utilization of sophisticated barrier techniques requires conscientious caretakers with extensive training and an excellent understanding of infection control concepts. Highly trained animal caretakers are essential for the animal facility of the future to minimize the entry of animal pathogens and to contribute toward decisions relating to diagnosis, containment, and elimination of animals exhibiting abnormal behaviors or signs of illness.

General Public

To complement the highly trained team of animal care professionals described above, scientists and others must educate the general public regarding the essential role of animal-based research in the growth of biomedical knowledge and continued improvements in health care. Surveys have shown that although most members of the public support the appropriate use of animals to study mammalian biology and human diseases, the level of knowledge is inadequate to defend against the well-funded, well-organized campaigns being waged against animal experimentation. Anti-animal research activists now boast that they contact children in 100% of the public and private elementary schools (Pacheco 1997). The information they present as fact is often unsubstantiated propaganda and falsehoods. At the same time, the urbanization of America has led to a larger proportion of the populace with little contact with animals used for food and fiber. Today's Americans are frequently exposed only to pet animals having "near family" status. Because a reversal in these trends is unlikely, the use of animals could be threatened by a caring but uneducated public.

For research to have a secure future, it is important for the public to comprehend the essential role played by animal-based research. Scientists at all levels must educate the public about the vital role of research animals and the safeguards that ensure their humane care and use. Inroads by animal activists have already increased the costs of animal research and have slowed research progress by ensnaring scientists and veterinarians in a veritable sea of regulatory red tape. Only an educated public can reverse these trends, provide tomorrow's scientists, and devote the funds necessary to promote the scientific progress that will improve human health into the 21st century.

INFRASTRUCTURE PROVISIONS

The infrastructure needed to support tomorrow's research animals will provide adequate protective housing, phenotyping and diagnostic imaging support, preservation of unique germplasm, rederivation of infected/infested animals, miniaturized telemetry and drug delivery devices, and specialized resources for studying nonhuman primate models. This infrastructure will require substantial and sustained investment and a clear vision of the future. We discuss the following aspects of infrastructure below: housing facilities, transgenic rodent resources, miniaturized equipment, monoclonal antibody resources, and regional primate resources.

Housing Facilities

Many research animal facilities were built 30 to 40 yr ago when various types of larger animals were used extensively in research. These facilities are no longer appropriate for today's highly defined, potentially immunodeficient research rodents and nonhuman primates. Both our knowledge of animal diseases and the standards for housing conditions have changed. Even though improved housing technologies partially compensate under some circumstances, numerous facilities not only fail to provide the protected environment required to sustain rodent barrier facilities but also lack the following: specialized areas such as Biosafety Level-3 housing, adequate ventilation, adequate thermal control, well-designed floor plans and room designs, caging that allows for environmental enrichment, and modern cage-washing facilities. The need for housing explosively growing populations of highly susceptible, valuable mice is a critical problem at a large number of leading institutions, and this problem will soon reach a critical level at many others (NCRR 1997) . A major reinvestment in infrastructure is required to prevent housing limitations from continuing to restrain scientific advancement. Although research institutions will bear the major burden of funding construction and renovation costs, matching federal funds and private foundation contributions could accelerate modernization. Expenditures for both renovation and construction must be accompanied by additional expenditures for modern protective caging and cage changing equipment such as change stations or biosafety cabinets, which comprise an integral part of the protective housing system for rodents. Caging provided for nonhuman primates should facilitate rather than impede environmental enrichment.

Transgenic Rodent Resources

As science has become global, it has become feasible to gain economy of scale from regionalized development of transgenic rodent resources. This financial approach is especially true for the expensive and specialized auxiliary resources required at the time of this writing to create, analyze, and preserve genetically altered rodents. Although most researchers throughout the country need access to embryo and embryonic stem cell injection facilities, phenotyping resources, rederivation services, and embryo and sperm cryopreservation services, only select commercial organizations and the largest of research institutions currently provide the full range of these services. Complete characterization of rodent models requires knowledge about murine development, murine geriatrics, murine behavior, and cognitive assessment. Fostering this knowledge will enable comprehensive evaluation of rodents from conception to death. Access to these services is essential to make full use of current animal models, regardless of an institution's size or location. The physical facilities and trained personnel required to provide the full range of these services are expensive and scarce.

Federal funding would appropriately ensure ready access to the resources described above by stimulating the development of regional resources with a mission to serve the scientific community at large, rather than limiting access to an elite cadre. Professional staff for such resources would ideally include microbiologists and geneticists who would play a key role in advancing diagnostic techniques and genetic monitoring. Regional laboratories also would be expected to take a lead role in training future specialists and in aiding investigators' attempts to dissect the effects of induced genetic alterations from the effects of exogenous factors such as environmental factors and infectious agents. These laboratories would similarly be important sites for additional research on improving and standardizing techniques for model creation and preservation.

Miniaturized Equipment

Miniaturized equipment will enable us to utilize fully the rodent and zebrafish models of the future (NRC 1998). Although some pieces of equipment, such as miniaturized telemetry devices and Alzet R® implantable osmotic pumps, are currently available in sizes suited to rodents, equipment availability limits the usefulness of some rodent and zebrafish models. Desirable equipment would include microsurgery tools and mechanisms for fluid sampling and drug delivery by various routes. Also needed are technological advances that allow us to assess the chemistry and physiology of individual cells in awake animals (NRC 1998).

Monoclonal Antibody Resources

Just as transgenic resources could benefit from regionalization, so could resources for monoclonal antibody production. At the time of this writing, in vitro methods are being promoted as humane alternatives to mouse ascites production, and many institutions are developing core monoclonal production facilities; however, most of these institutional facilities lack the work volume to benefit from methods providing economies of scale. In addition, current in vitro technology does not permit investigators to produce varying quantities of all the necessary monoclonals in a cost-efficient manner. Thus, investigators have often resisted moving to in vitro techniques because cost efficiency, volume, and quality (titer and avidity) are virtually impossible to predict. Regional monoclonal production facilities would provide the advantage of specialists who could make scientific advances in the field while ensuring that required amounts of each monoclonal antibody would be produced in ways that were both humane and cost efficient.

Regional Primate Resources

Regional primate centers are 1 example of how regional resources can provide access to expensive and scarce animals and can promote research progress more efficiently than institution-based resources. We expect that nonhuman primates will continue to serve as crucial animal models because of their unique similarities to humans, and we anticipate the continued development of regional primate resources. Although we can learn about basic mammalian biological processes from genetically manipulated rodents, understanding primate biological processes will require studies using nonhuman primates or humans. Whereas many different primate species have been used during the 20th century, a narrower range of species is expected to be key to studies in the 21st century. Those species will include Macaca fascicularis (cynomolgus monkeys), Macaca nemestrema (pigtailed macaque), Macaca mulatta (Rhesus monkeys), Papio sp. (baboons), Saimiri sp. (squirrel monkeys), Callithrix sp. (marmosets), and, for selected infectious disease models for which no other species can be substituted, Pan troglodytes (chimpanzees). Nonhuman primates used in the 21st century, like rodents being used at the time of this writing, will be largely purpose bred in domestic facilities and will be free of endemic infection with adventitious such as Circopithecine herpesvirus 1 (formerly Herpesvirus simiae, B virus) and simian retroviruses.

SUMMARY

We believe that trained individuals---veterinarians, scientists, animal care staff, and the general public---are central to continued growth in biomedical knowledge. The efforts of these people must be supported by modernized infrastructures---animal housing facilities, transgenic rodent resources, miniaturized equipment, antibody resources, and primate resources---for us to continue to improve health care and gain biomedical knowledge in the 21st century utilizing animal models.

ACKNOWLEDGMENT

We thank Dr. Abigail L. Smith, Loyola University, Chicago, Illinois, for helpful editorial comments.

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