4 Health Surveillance Programs

Health surveillance (or health 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 tests for the purpose of defining the pathogen and health status of an animal population. Health surveillance programs are crucially important in rodent disease prevention-they provide data, the only reliable basis for rodent pathogen status or 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, 1984). No two programs are identical. Some are limited in scope, while others are very comprehensive. Numerous factors should be considered in designing individual programs, with special emphasis placed on objectivity in testing rather than merely 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 requirements to meet the specific



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--> 4 Health Surveillance Programs Health surveillance (or health 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 tests for the purpose of defining the pathogen and health status of an animal population. Health surveillance programs are crucially important in rodent disease prevention-they provide data, the only reliable basis for rodent pathogen status or 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, 1984). No two programs are identical. Some are limited in scope, while others are very comprehensive. Numerous factors should be considered in designing individual programs, with special emphasis placed on objectivity in testing rather than merely 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 requirements to meet the specific

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--> scientific objectives of individual research programs. Clearly, the requirements for a study of transmissible leukemia will differ from those of a study of chemical carcinogenesis of the respiratory tract in mice. Similarly, an elaborate program may be required to ensure the validity of research results from a project comparing the immune responses of immunodeficient mouse strains to challenge with an infectious agent, whereas a modest program might suffice for a study concerned with inheritance of a coat color gene in mice. Just as research objectives can differ greatly, health surveillance requirements also may vary over a wide range. Agent Detection Objectives Indigenous infections of laboratory rodents include strong pathogens, weak pathogens, opportunists, and commensals. Therefore, in designing health surveillance programs, decisions must be made as to which agents are to be covered in the test battery. 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. Therefore, testing is, of necessity, always selective. Specific justification based on pathogen significance and likelihood of interference with research is needed to include an agent in the test battery. 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 of these may include very 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 (Table 7). In recent years 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. The ELISA and IFA tests have much greater sensitivity than the CF and HAI tests, and they give much fewer false positives than the HAI test (Smith, 1986b). False positive HAI tests have been particularly troublesome in the diagnosis of reovirus-3 and Theiler's virus infections (Kraft and Meyer, 1986; Van Der Logt, 1986). Serologic tests have the advantages of being relatively inexpensive and quickly performed in comparison to virus isolation (Balk, 1983; Smith, 1986b).

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--> TABLE 7 Serologic Tests for Detection of Infectious Agents in Mice and Ratsa Agent Recommended Restsb Tests Not Recommended Speciesc Comments A. Virusesd         Adenoviruses ELISA, CF   M, R Test should include both FL and K87 antigens. Cytomegalovirus ELISA   M, R   Hantaviruses IFA, NT, HAI   R   Kilham rat virus ELISA, HAI   R   K virus HAI   M Value of test for rats is uncertain. Lactic dehydrogenase virus Lactic dehydrogenase assay   M Serum must be kept frozen to preserve enzyme activity. LCMV IFA, ELISA CF M, R   Mousepox virus ELISA, IFA CF. HAI M   Minute virus of mice ELISA, IFA   M   Mouse hepatitis virus ELISA, IFA CF M Tests have only coronavirus specificity. Mouse rotavirus ELISA   M   Mouse thymic virus IFA   M Test not available commercially. Pneumonia virus of mice ELISA, HAI, IFA   M, R   Polyoma virus ELISA, HAI   M   Reovirus-3 ELISA, IFA   M   Sendai virus ELISA, HAI, CF, IFA   M, R   SDA/RCV ELISA, IFA CF R Tests have only coronavirus specificity. Theiler's virus ELISA, NT HAI M Value of tests for rats is uncertain. Toolan H-1 virus ELISA, HAI   R   B. Bacteria         CAR Bacillus ELISA   R   Corynebacterium kutscheri ELISA   M, R   Bacillus piliformis CF, IFA   M, R Tests not available commercially. Leptospira interrogans IFA   M, R Test available through state animal diagnostic laboratories. Mycoplasma arthritidis ELISA CF M, R Test has only Mycoplasma genus specificity. Mycoplasma pulmonis ELISA CF M, R Test has only Mycoplasma genus specificity. C. Protozoa         Encephalitozoon cuniculi IFA and others   M, R   ª For further information and references, refer to summaries of individual agents in Part II of this volume. b Abbreviations used for tests: ELISA = enzyme-linked immunosorbent assay, CF = complement fixation, IFA = indirect immunofluorescence, NT = neutralization test, HAI = hemagglutination inhibition. c M = mouse; R = rat. d Modified from information originally compiled by P.N. Bhatt (Section of Comparative Medicine, Yale University. New Haven, Conn.).

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--> Serologic tests also have certain disadvantages. They require special technical competence to assure rigorous standardization of reagents, the inclusion of appropriate controls in each test run and accurate interpretation of results (Smith, 1986b). They are indirect tests that rely on humoral antibody responses, and they vary in specificity and sensitivity, depending on the test and the agent. Therefore, positive results based on a single serologic procedure are far less definitive in diagnostic value than positive results of a direct test such as isolation and identification of an agent. For this reason, serologic testing should rely on a primary test for each agent and one or more tests to confirm the positive results of any primary test (Smith, 1986b). False-positive test results can occur on serum from young animals due to passively acquired antibody. False-negative results may be obtained on serum from young animals infected prior to the age of immunocompetence or on serum from animals infected for too brief a time for an antibody response to be mounted (Jacoby and Barthold, 1981). One of the most useful applications of serologic testing in rodent health surveillance is the mouse antibody production (MAP) test. Although originally developed as a method for broadly screening mouse tissues for viruses (Rowe et al., 1959a, 1962), it can be used to test transplantable tumors, hybridomas, cell lines, and other biologic materials for contaminating infectious agents of different types. As originally performed by Rowe et al. (1959a, 1962), test material is injected intraperitoneally into a group of five pathogen-free mice; they are maintained in a caging system that excludes pathogen contamination for five weeks, then killed and individually tested serologically for a range of agents. Uninoculated, separately housed control mice must be used (Rowe et al., 1959a, 1962). An equivalent test procedure, the rat antibody production (RAP) test, can be done using rats (Johnson, 1988). In general the MAP or RAP test is considered more sensitive than virus isolation (de Souza and Smith, 1989). The isolation of bacteria by cultural methods and the demonstration and identification of parasites are the standard procedures for the detection of these agents. However, these methods also have limitations that depend 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 site(s) and processed expeditiously by methods known to maximize the chances of successful recovery or demonstration of the agent. Gross and microscopic evaluations of tissues for lesions are also invaluable in health surveillance. In the more comprehensive health surveillance programs, histopathologic examination of all major organs by a qualified

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--> pathologist is standard practice. Lesions due to pathogen infections can occur before seroconversion in viral infections. Some histopathologic changes are diagnostic, and others provide clues of disease processes for further investigations (Jacoby and Barthold, 1981). The foregoing discussion has presented the major methodological approaches presently in use in rodent diagnostic laboratories. It must be recognized, however, that diagnostic methodology currently is in transition. Refinements continue to be made in existing methods and newer methods employing molecular biology, 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; Grody et al., 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 with two important assumptions: rates of infection and randomness in sampling (ILAR, 1976a; Hsu et al., 1980; Small, 1984; DiGiacomo and Koepsell, 1986). As shown in Table 8, if one assumes that 40% of the animals in a population are infected with an agent, there is a 99% probability that one infected animal will be detected in a randomly selected sample of 10 animals. At a 50% infection rate, a sample size of only five is required for a 97% probability of detecting infection in at least one animal. It should be recognized that although the sample size required to detect a single agent can be determined with reasonable precision (Table 8), 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. A health surveillance test battery for these three agents, then, would be required to use the lower assumed infection rate (i.e., 5%); 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. Proper sampling also requires random sampling of the entire population. This means taking animals from different cages, shelves, and racks. Attention

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--> TABLE 8 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                     ª From ILAR (1976a), Hsu et al. (1980), and Small (1984). b N = log(l - probability of detecting infection) log(l - assumed infection rate) also should be given to sampling animals of both sexes and of different ages. Sampling of two age groups is desirable. Young animals tend to have greater parasite burdens. 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 indication of the infection history of the colony (Jacoby and Barthold, 1981). Test Frequency One of the most difficult questions to be answered in designing health surveillance programs is how frequently should a given rodent population 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 a pathogen or other contamination is to the use of the population, the level of risk of pathogen contamination from other nearby rodent populations, and economic considerations. After evaluating these basic questions, one should have a basis for deciding whether testing should be monthly, quarterly, biannually, or annually. However, the

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--> 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 the larger battery could be done biannually. Sentinel Animals Rodents for health surveillance purposes are sometimes introduced into a rodent population, housed in open cages placed systematically throughout the colony, and designated as sentinels for use in periodic testing. Pathogen transmission from the principal population to the sentinels may 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 stock as the principal population and should be subjected to any experimental treatments given the principal population. The introduction of a second stock as sentinels, although tested and found to be free of pathogens, may pose an unnecessary risk for contaminating the principal population. Rodent Diagnostic Laboratories Rodent diagnostic laboratories are indispensable to the production and maintenance 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 expertise may also be required in some instances. Many of the larger research institutions have well-equipped and -staffed institutional diagnostic laboratories. Testing services also can be obtained through commercial laboratories. Traditionally, rodent diagnostic laboratories have tended to give highest priority to the investigation of clinical illnesses and necropsy evaluations of dead animals. That approach is no longer acceptable. While those services are certainly necessary, the needs of modern research and the principles of the scientific method demand that diagnostic laboratories give greater priority to rodent disease prevention. Most of the pathogen infections and pathogen-induced diseases of laboratory rodents are preventable.

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