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PART I Principles of Rodent Disease Prevention SCIENTIFIC OBJECTIVES Animal experiments are essential to progress in the biomedical sciences (NRC, 1985~. Like investigations in any field of science, the merit of animal experiments ultimately depends on rigid adherence to the principles of scientific method. Proper practice of these principles yields data that are both reliable and reproducible, key objectives of all good experiments (Bernard, 1865~. INFECTION VERSUS DISEASE A common misconception is that infection is synonymous with disease. Bacte- rial opportunists and commensals, which are constitutents of the normal flora on mucosal and body surfaces, are ubiquitous infections that usually cause disease only when their hosts are immunosuppressed (Dubos et al., 1965; Savage, 1971~. The viral and parasite pathogens of rodents vary considerably in pathogenicity. Some cause severe disease, while others rarely do. It is also important to distinguish between subclinical (inapparent, covert, or silent) and clinically apparent infec- tions. Most natural infections with pathogenic organisms in mice and rats are subclinical, and infection-induced aberrations in research results often occur in the absence of clinical disease. Thus, it is important to prevent infection, not merely to prevent clinical disease. 1

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2 COMPANION GUIDE TO INFECTIOUS DISEASES OF MICE AND RATS TERMINOLOGY OF MICROBIAL AND PATHOGEN STATUS Terms used in defining rodent microbial status vary greatly in precision of meaning. Four terms (germfree, gnotobiotic, defined flora, and conventional), representing the extremes of microbial status, have clear definitions that are generally accepted and understood by scientists, as well as by technical personnel (NRC, 1991~. However, there is major confusion about the definition and use of terms representing the middle ground of pathogen status. Pathogen free, specific pathogen free, virus antibody free, and clean conventional are relative terms that require explicit definition every time they are used. The definition should include the background of the rodent subpopulation in question (e.g., cesarean derived, isolator maintained, barrier maintained), details of current housing (e.g., isolator, barrier), and data from laboratory tests for pathogens (the specific tests done, the number of tests, the frequency of testing, and the results) (Lindsey et al., 1986~. COMMITMENT TO MAINTAINING PATHOGEN-FREE STATUS OF RODENTS Past experience demonstrates that maintaining rodents in the pathogen-free state requires adherence to breeding, transportation, and maintenance programs spe- cifically designed for the exclusion of pathogens. This means a strong commit- mentby investigators, research staff, and animal care staff. Some essential elements of that commitment are as follows:* a. The investigator and the support personnel must understand the terminology and principles involved. b. Appropriate facilities and equipment must be available. c. Housing practices must ensure physical separation and avoidance of cross- contamination between different animal subpopulations throughout their lives. d. Reliable health monitoring should be maintained to identify breeding popu- lations free of pathogens and to redefine the microbiologic status of the animals at regular intervals from the time they are received in the user facility until completion of each study. e. Written standard operating practices must be developed and followed without interruption; clear objectives must be defined in advance, along with detailed procedures for reaching those objectives. *From a consensus developed during a seminar entitled "B. arrier Maintenance of Rodents in Multipurpose Facilities," held at the Thirty-Sixth Annual Session of the American Association for Laboratory Animal Science on November 3-8, 1985, in Baltimore, Md. Participants were J. R. Lindsey (leader); G. L. Van Hoosier, Jr.; D. B. Casebolt; J. G. Fox; R. O. Jacoby; and T. E. Hamm, Jr.

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PRINCIPLES OF RODENT DISEASE PREVENTION 3 HEALTH SURVEILLANCE PROGRAMS Health surveillance (or monitoring) is the term usually applied to the testing of laboratory animals to determine their pathogen status and general state of health. Health surveillance programs are systematic laboratory investigations that employ batteries of diagnostic tests for the purpose of defining the pathogen and health status of an animal population. These programs are crucially important in rodent disease prevention because they provide data, which are the only reliable basis for determining rodent pathogen status or providing health quality assurance. Although the need for health surveillance programs is generally accepted, there is a great diversity of opinion about the design of individual programs (Hsu et al., 1980; Iwai et al., 1980; Thigpen and Tortorich, 1980; Jacoby and Barthold, 1981; Hamm, 1983; Loew and Fox, 1983; Small, 19841. No two programs are identical. Some are limited in scope; others are very comprehensive. Numerous factors should be considered in designing individual programs, and special emphasis should be placed on objectivity in testing rather than on the adoption of customary practices. Some of those factors are listed in the following sections. Scientific Objectives Health surveillance efforts should, to the fullest extent possible, be matched qualitatively and quantitatively with the specific scientific objectives of individual research programs to ensure that the quality of the animal will meet these objectives. For practical reasons, it is impossible to test for all known infectious agents of rodents, or even all infectious agents that theoretically could interfere with a particular study. In designing health surveillance programs, decisions must be made about which agents should be covered in the test battery. Inclusion of a pathogen in the test battery should be based on the likelihood that it will interfere with the research being conducted. Such information is given in Part II of this volume. Test Procedures The procedures used in health surveillance generally include serologic tests, bacterial cultures, parasitologic examinations, and histopathology. Each category can include a few or many procedures to detect different infectious agents or disease processes. Some health surveillance programs are limited to only one of these types of procedures, e.g., serologic testing. Serologic tests are the main procedures used for detecting virus infections in rodents, but they also have been found useful for some bacterial and protozoan infections. The enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescent antibody (IFA) test have largely replaced the complement fixation (CF) test and the hemagglutination inhibition (HAI) test. They are much more sensitive than either the CF or HAI test and give fewer false positives than the

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4 COMPANION GUIDE TO INFECTIOUS DISEASES OF MICE AND RATS HAI test. Serologic testing should rely on a primary test for each agent and one or more additional tests to confirm the positive results of any primary test (Kraft and Meyer, 1986; Smith, 1986b; Van Der Logt, 1986~. One of the most useful applications of serologic testing in rodent health surveillance is the mouse antibody production (MAP) test (Rowe et al., 1959, 1962~. Although originally developed as a method for broadly screening mouse tissues for viruses, it can be used to test transplantable tumors, hybridomas, cell lines, and other biologic materials for contamination by infectious agents. An equivalent test, the rat antibody production (RAP) test, is useful for screening biologic materials taken from rats. Both these tests are generally considered more sensitive than virus isolation (de Souza and Smith, 19891. The isolation of bacteria using cultural methods and the demonstration and identification of parasites using a microscope are the standard procedures for detection of these agents. However, these methods also have limitations, depending on the agent. In general, causative agents are more difficult to isolate or demonstrate in subclinical infections than in clinically apparent infections. Some bacterial infections of mice and rats, e.g., Corynebacterium kutscheri or Mycoplasma arthritidis, commonly occur as subclinical infections in which cultural isolation is extremely difficult. With each of the bacteria and parasites it is imperative that specimens be collected from the most appropriate siteks) and processed expedi- tiously using methods known to maximize the chances of successful recovery or demonstration of the agent. Failure to collect specimens from the site that is most appropriate for that microbe can result in false-negative tests. Gross and microscopic evaluations of tissues for lesions are also invaluable in health surveillance. In more comprehensive health surveillance programs, histo- pathologic examination of all major organs by a qualified pathologist is standard practice. Lesions caused by viral pathogens can occur before seroconversion. Some histopathologic changes are diagnostic; others provide only clues to disease processes. Diagnostic methodology is in transition. Refinements continue to be made in existing methods, and newer methods employing molecular biologic techniques, e.g., nucleic acid hybridization and specific gene product detection, are being developed at a rapid pace (Sklar, 1985; Edberg, 1986; Smith, 1986a,c; Delellis and Wolfe, 1987; Howanitz, 1988). Sampling Strategies The purpose of health surveillance is to detect at least one animal with each of the infections or diseases present in the population. The purpose is not to determine prevalence of infection or disease. The number of animals (sample size) to be tested is of critical importance and can be determined mathematically by making important assumptions about the rates of infection and the randomness in sampling (ILAR, 1976; Hsu et al., 1980; Small,

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PRINCIPLES OF RODENT DISEASE PREVENTION 5 TABLE 1 Confidence Limits for Detecting Infection Using Different Sample Sizes and Assumed Rates of Infectiona ... . . Sample Assumed Infection Rate (%) Size (N)b 1 2 3 4 5 10 15 20 25 30 40 50 5 0.05 0.10 0.14 0.18 0.23 0.41 0.56 0.67 0.76 0.83 0.92 0.97 10 0.10 0.18 0.26 0.34 0.40 0.65 0.80 0.89 0.94 0.97 0.99 15 0.14 0.26 0.37 0.46 0.54 0.79 0.91 0.95 0.99 20 0.18 0.33 0.46 0.56 0.64 0.88 0.95 0.99 25 0.22 0.40 0.53 0.64 0.72 0.93 0.98 30 0.25 0.45 0.60 0.71 0.79 0.96 0.99 35 0.30 0.51 0.66 0.76 0.83 0.97 40 0.33 0.55 0.70 0.80 0.87 0.99 45 0.36 0.69 0.75 0.84 0.90 0.99 50 0.39 0.64 0.78 0.87 0.92 0.99 60 0.45 0.70 0.84 0.91 0.95 70 0.51 0.76 0.88 0.94 0.97 80 0.55 0.80 0.91 0.96 0.98 90 0.60 0.84 0.94 0.97 0.99 100 0.63 0.87 0.95 0.98 0.99 120 0.70 0.91 0.97 0.99 140 0.76 0.94 0.99 160 0.80 0.96 0.99 180 0.84 0.97 200 0.87 0.98 aFrom ILAR (1976a), Hsu et al. (1980), and Small (1984). bat _ log(1 - probability of detecting infection) log(1 - assumed infection rate) 1984; DiGiacomo and Koepsell, 1986~. As shown in Table 1, if one assumes that 40% of the animals in a population are infected with an agent, there is a 99% probability that 1 infected animal will be detected in a randomly selected sample of 10 animals. At a 50% infection rate, a sample size of only 5 is required for a 97% probability of detecting infection in at least 1 animal. Although the sample size required to detect a single agent can be determined with reasonable precision, it is virtually impossible to maintain the same degree of precision for all agents to be included in a large test battery. Different agents typically have very different infection rates within rodent colonies. For example, typical rates for established infections in mouse colonies are greater than 90% for Sendai virus, approximately 25% for pneumonia virus of mice, and less than 5% for Salmonella enteritidis. In determining the number of animals to be used in a health surveillance test battery for these three agents, the lowest assumed infection rate should be used (i.e., 5%), and a 95% confidence limit would require a sample size of at least 60 animals. This is entirely appropriate in instances where subclinical S. enteritidis infection is suspected. However, for routine health surveillance, sample sizes are usually based on assumed infection rates of 40-50% in order to keep sample sizes reasonable.

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6 COMPANION GUIDE TO INFECTIOUS DISEASES OF MICE kD RATS Proper sampling also requires that animals be taken from different cages, shelves, and racks so that the sample is representative of the entire population. Animals of both sexes and of two age groups should be sampled. For serologic testing, sampling of young adults (approximately 90 days old) and retired breeders is recommended. Young adults are best for detecting recent viral infections (without interference from passive antibody), and retired breeders give an indica- tion of the colony's infection history (Jacoby and Barthold, 1981~. Test Frequency One of the most difficult decisions to be made in designing health surveillance programs is how frequently a given rodent population should be tested. There are no established guidelines, but the problem seems to revolve around four central issues: the specific purpose of the population in question, the potential or real importance of a pathogen or other contamination to use of the population, the level of risk of pathogen contamination from other nearby rodent populations, and economic considerations. After evaluating these issues, one should have a basis for deciding whether testing should be monthly, quarterly, biannually, or annually. However, the frequency of testing may be different for different agents. For example, if the greatest risks are deemed to be from mouse hepatitis virus and Sendai virus, tests for these agents could be performed monthly, and a larger battery could be done biannually. Sentinel Animals Sentinel rodents are sometimes introduced into a rodent population, housed in open cages placed systematically throughout the colony, and used periodically for testing. Pathogen transmission from the principal population to the sentinels can be increased by transferring the sentinels into dirty cages from the principal population at each cage change. Sentinel animals preferably should be of the same population as the principal population and should be subjected to any experimental treatments given to the principal population. The introduction of a second population as sentinels, even if it is tested and found to be free of pathogens, poses an unnecessary risk for contaminating the principal population. RODENT DIAGNOSTIC LABORATORIES Rodent diagnostic laboratories are indispensable to the production and mainte- nance of mice and rats for high-quality research. Such laboratories specialize in health surveillance testing, investigations of clinical diseases, and other quality control methods specifically designed for laboratory rodents. Depending on the breadth of their activities, these laboratories most often include competence in serology, bacteriology, parasitology, and pathology. Virology and hematology

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PRINCIP f ES OF RODENT DISEASE PREVENTION 7 expertise may also be required in some instances. Many larger research institutions have well-equipped and well-staffed institutional diagnostic laboratories. Testing services also can be obtained through commercial laboratories. Traditionally, rodent diagnostic laboratories have tended to give highest Snooty to the investigation of clinical illnesses and necropsy evaluations of dead animals. That approach is no longer acceptable. While those services certainly are necessary, the needs of modern research and the principles of scientific method demand that diagnostic laboratories give greater priorly to disease prevention. Most of the pathogen infections and pathogen-induced diseases of laboratory rodents are preventable. REFERENCES Bernard, C. 1865. An Introduction to the Study of Experimental Medicine (English translation byH.C.Greene,1927~. New York: MacMillan. 226 pp. de Souza, M., and A. L. Smith. 1989. Comparison of isolation in cell culture with conventional and modified mouse antibody production tests for detection of murine viruses. J. Clin. Microbiol. 27:185-187. DeLellis, R. A., and H. J. Wolfe. 1987. New techniques in gene product analysis. Arch. Pathol. Lab. Med. 111:620-627. DiGiacomo, R. F., and T. D. Koepsell. 1986. Sampling for detection of infection or disease in animal populations. J. Am. Vet. Med. Assoc. 189:22-23. Dubos, R. J., R. W. Schaedler, R. Costello, and P. Hoet. 1965. Indigenous, normal and autochthonous flora of the gastrointestinal tract. J. Exp. Med. 122:67-82. Edberg, S. C. 1986. Nucleic acid hybridization analysis to elucidate microbial pathogens. Lab. Med. 17:735-738. Hamm, T. E., Jr. 1983. The effects of health and health monitoring on oncology studies. Pp. 45-60 in The Importance of Laboratory Animal Genetics, Health and the Environment in Biomedical Research, E. C. Melby, Jr., and M. W. Bald, eds. New York: Academic Press. Howanitz, J. H. 1988. Immunoassay. Arch. Pathol. Lab. Med. 112:771-779. Hsu, C. K., A. E. New, and J. G. Mayo. 1980. Quality assurance of rodent models. Pp.17- 28 in Animal Quality and Models in Biomedical Research, A. Spiegel, S. Erichsen, and H. A. Solleveld, eds. Stuttgart: Gustav Fischer Verlag. ILAR (Institute of Laboratory Animal Resources). 1976. Long-term holding of laboratory rodents. A report of the Committee on Long-Term Holding of Laboratory Rodents. ILAR News l9:L1-L25. Iwai, H., T. Itch, N. Nagiyama, and T. Nomura. 1980. Monitoring of murine infections in facilities for animal experimentation. Pp. 219-222 in Animal Quality and Models in Biomedical Research, A. Spiegel, S. Erichsen, and H. A. Solleveld, eds. Stuttgart: Gustav Fischer Verlag. Jacoby, R. O., and S. W. Barthold. 1981. Quality assurance for rodents used in toxicological research and testing. Pp.27-55 in Scientific Considerations in Monitoring and Evaluating Toxicological Research, E. J. Gralla, ed. Washington, D.C.: Hemisphere. Kraft, V., and B. Meyer. 1986. Diagnosis of murine infections in relation to test method employed. Lab. Anim. Sci. 36:271-276.

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8 COMPANION GUIDE TO INFECTIOUS DISEASES OF MICE AND RATS Lindsey, J. R., D. B. Casebolt, and G. H. Cassell. 1986. Animal health in toxicological research: An appraisal of past performance and future prospects. Pp. 155-171 in Managing Conduct and Data Quality of Toxicology Studies. Princeton, N.J.: Princeton Scientific. Loew, F. M., and J. G. Fox. 1983. Animal health surveillance and health delivery systems. Pp. 69-82 in The Mouse in Biomedical Research. Vol. III: Normative Biology, Immunology, and Husbandry, H. L. Foster, J. D. Small, and J. G. Fox, eds. New York: Academic Press. NRC (National Research Council). 1985. Models for Biomedical Research: A New Perspective. A report of the Board on Basic Biology, Committee on Models for Biomedical Research. Washington, D.C.: National Academy Press. 180 pp. NRC (National Research Council). 1991. Infectious Diseases of Mice and Rats. A report of the Institute of Laboratory Animal Resources Committee on Infectious Diseases of Mice and Rats. Washington, D.C.: National Academy Press. 397 pp. Rowe, W. P., J. W. Hartley, J. D. Estes, and R. J. Huebner. 1959. Studies of mouse polyoma virus infection. I. Procedures for quantitation and detection of virus. J. Exp. Med. 109:379-391. Rowe, W. P., J. W. Hartley, and R. J. Huebner. 1962. Polyoma and other indigenous mouse viruses. Pp.131-142 in The Problems of Laboratory Animal Disease, R. J. C. Harris, ed. New York: Academic Press. Savage, D. C. 1971. Defining the gastrointestinal microflora of laboratory mice. Pp.60-73 in Defining the Laboratory Animal. Proceedings of the Fourth International Symposium on Laboratory Animals, organized by the International Committee on Laboratory Animals and the Institute of Laboratory Animal Resources and held April 8-11, 1969, in Washington, D.C. Washington, D.C.: National Academy of Sciences. Sklar, J. 1985. DNA hybridization in diagnostic pathology. Human Pathol. 16:654-658. Small, J. D. 1984. Rodent and lagomorph health surveillance~uality assurance. Pp.709- 723 in Laboratory Animal Medicine, J. G. Fox, B. J. Cohen, and F. M. Loew, eds. New York: Academic Press. Smith, A. L. 1986a. Detection methods for rodent viruses. Pp. 123-142 in Complications of Viral and Mycoplasmal Infections in Rodents to Toxicology Research and Testing, T. E. Hamm, Jr., ed. Washington, D.C.: Hemisphere. Smith, A. L. 1986b. Serologic tests for detection of antibody to rodent viruses. Pp.731 -751 in Viral and Mycoplosmal Infections of Laboratory Rodents: Effects on Biomedical Research, P. N. Bhatt, R. O. Jacoby, H. C. Morse III, and A. E. New, eds. Orlando, Fla.: Academic Press. Smith, A. L. 1986c. Methods for potential application to rodent virus isolation and identification. Pp.753-776 in Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research. P. N. Bhatt, R. O. Jacoby, H. C. Morse III, and A. E. New, eds. Orlando, Fla.: Academic Press. Thigpen, J. E., and J. A. Tortorich. 1980. Recommended goals for microbiological quality control for laboratory animals used in the National Toxicology Program (NTP). Pp. 229- 233 in Animal Quality and Models in Biomedical Research, A. Spiegel, S. Erichsen, and H. A. Solleveld,eds. Stuttgart: Gustav Fischer Verlag. Van Der Logt, J. T. M. 1986. Serological study on the prevalence of murine viruses in laboratory animal colonies, in France and in the Netherlands (1981-19841. Sci. Tech. Anim. Lab.: 11 :195-203.