Microbial and Phenotypic Definition of Rats and Mice: Proceedings of the 1998 US/Japan Conference (1999)

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Current Status of Pathogen Status in Mice and Rats J. Russell Lindsey Professor, Department of Comparative Medicine University of Alabama Schools of Medicine and Dentistry Birmingham, Alabama Following the pattern set by the previous Japanese speakers, Drs. Itoh and Mannen, Dr. Weisbroth has addressed the issue of “testing methods,” and I will speak on “current status” of pathogens in mice and rats. I am, of course, most familiar with the quality of animals in my own country, the United States. How- ever, many of my comments will be applicable worldwide as biomedical research and its constituent parts, including the laboratory animals being used to generate much of the data and the journals that disseminate the results, are increasingly international activities. PAST PROGRESS TOWARD REDUCING PATHOGENS Much progress has been made in eliminating pathogens from laboratory mouse and rat populations since the 1960s (Weisbroth 1996). This fact is borne out by several surveys conducted mainly in the early 1980s (NRC 1991, pp. 7-8) and one in 1988 (Casebolt and others 1988). During the period from the 1960s into the 1990s, there were also major efforts to (1) improve methods for detect- ing, eliminating, and preventing pathogen infections (Allen and Nomura 1986; Baker 1998; Bhatt and others 1986; NRC 1991); and (2) herald the seemingly endless detrimental effects that pathogens have on research results (Allen and Nomura 1986; Bhatt and others 1986; NRC 1991 [see partial listings on pp. 274- 275]). More recently, many additional examples of pathogen effects have been published (Baker 1998). Although all of these past developments are noteworthy achievements that tend to engender a great sense of accomplishment among laboratory animal specialists, it seems to me far more important to take the 39 

40 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE forward view and ask the question, “How well are these achievements serving contemporary research?” The answers to this question are rather disappointing for a number of reasons. PATHOGENS STILL POSE PERVASIVE RISKS IN THE UNITED STATES Dr. Jacoby and I recently surveyed the top 100 institutional recipients of National Institutes of Health (NIH) funds regarding their efforts to prevent patho- gen infections in mouse and rat populations at their institutions. Responses were received from 72 of the 100 institutions, representing more than $5 billion in NIH support and use of 3 million mice and 1 million rats for the year of the survey (Jacoby and Lindsey 1997, 1998). The results were surprising. Only 70% of the mouse populations and 60% of the rat populations were considered specific pathogen free (SPF), meaning that many populations were, in reality, conven- tional. Furthermore, the survey was constructed so that each institution could define its use of the term SPF by checking presence or absence of each of 24 pathogens. The results showed enormous disparities among institutions in the definition of SPF. Agents such as ectoparasites, parvoviruses, mouse hepatitis virus, mouse rotavirus, Helicobacter sp., Theiler’s encephalomyelitis virus, and cilia-associated respiratory bacillus, generally recognized as pathogens, were common in so-called SPF populations. There was also great variation in the frequency of testing for pathogens, the number of animals tested, and the test methods being used. These findings point to real problems with the terminology of pathogen status. PATHOGEN STATUS GETS LOST IN THE TERMINOLOGY MORASS Unfortunately, the hard reality is that pathogen status is elusive because it is usually defined by words selected from a morass (“a marsh, swamp, or something that traps, confuses, or impedes”) (Webster’s Ninth New Collegiate Dictionary 1989) of terminology, including words such as germ free, defined flora, pathogen free, specific pathogen free, virus antibody free, barrier maintained, and conven- tional. The underlying problem is that these terms merely identify concepts. The results of the terminology morass are (1) there is no universal testing strategy or reporting terminology for clear and consistent definition of pathogen status in rodent populations, and (2) each institution (or investigator) selects its own list of pathogens, test procedures, animal sampling strategy, frequency of sampling, and reporting terminology. Definitions of the conceptual terms above should include the agents for which tests were actually done on the subpopulation(s) in question, test methods used, results of the tests, and frequency of the testing. 

J. RUSSELL LINDSEY 41 PRINCIPLES OF PATHOGEN STATUS ARE BEING COMPROMISED Based on the foregoing information, it appears that a number of key prin- ciples of rodent pathogen status are being compromised: 1. Infectious agents that have been documented to cause significant disease or to interfere with research results need to be recognized as pathogens in the context of research use. 2. Pathogens of rodents are notorious for causing subclinical infections. 3. Many subclinical pathogen infections cause altered research results. 4. No meaningful inferences can be made about pathogen status without the benefit of results from tests for specific agents. 5. Immunodeficient animals require different test batteries than immuno- competent animals. 6. Tests for pathogens are performed on subpopulations of animals, not on “facilities.” Terms such as “pathogen free” are actually concepts and must be defined each time they are used (Allen and Nomura 1986; Bhatt and others, 1986; NRC 1991). Some of the reasons these principles are being compromised in the United States are as follows: 1. The NIH National Center for Research Resources (NCRR) supported diagnostic and investigational laboratories were phased out (NRC 1998). 2. The Office of Management and Budget cost accounting standards shifted payment for diagnostics to investigators (NRC 1998). 3. Regulatory issues are requiring increasing attention (NRC 1998). 4. Rodent populations are increasing dramatically (transgenics, immuno- deficients) (Jacoby and Lindsey 1997, 1998). 5. Transfers of animals between laboratories are increasing worldwide (Jacoby and Lindsey 1997, 1998). 6. Overt clinical disease is not widespread (all rodent pathogens tend to cause subclinical infections). 7. Investigators are becoming increasingly focused on molecular events and less aware of integrative biology. 8. There is little appreciation of pathogen importance among investigators, as evidenced by the following. SCIENTISTS HAVE LITTLE APPRECIATION OF PATHOGEN STATUS Since Dr. Jacoby and I published the results of the survey of institutions in the United States on their programs for protecting mouse and rat populations 

42 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE TABLE 1 Documentation of Pathogen Status in Scientific Articles Status defined PF,a SPF,a etc., No status Total papers Journal (volume) by test results used, not defined information reviewed Inf. Immun.a (65) 3 23 74 100 J. Immunol.a (158) 4 18 78 100 Am. J. Pathol.a (150-151) 3 16 81 100 P.N.A.S.a (94) 1 4 95 100 Science (275-280) 0 0 100 100 aAm. J. Pathol., American Journal of Pathology; Inf. Immun., Infection and Immunity; J. Immunol., Journal of Immunology; PF, pathogen free; P.N.A.S., Proceedings of the National Academy of Sciences U. S. A.; SPF, specific pathogen free. from pathogens (Jacoby and Lindsey 1997, 1998), I have often wondered what affect rodent health testing or the lack thereof has on the quality of the results appearing in scientific journals, and how investigators and journal editors per- ceive the importance of pathogen status. As a result, I chose five journals and reviewed in each of them the first 100 original scientific articles using mice or rats, beginning with their January 1997 issue. I searched each article for infor- mation defining pathogen status (Table 1). The journals were Infection and Immunity, Journal of Immunology, American Journal of Pathology, Proceedings of the National Academy of Sciences U. S. A., and Science. The results clearly indicate that investigators and journal editors have little appreciation of pathogen status. In summary, let me simply reiterate some of the main “take home” messages about the current status of pathogen status of mice and rats used in research. Although progress has been made over the past 40 years in reducing their preva- lence, rodent pathogens still present serious risks to a substantial amount of the research data being generated every year. Unfortunately, there are many obstacles to further reductions in pathogen prevalence, including problems of terminology, adherence to sound principles of pathogen exclusion, lack of financial support for health surveillance testing, and lack of appreciation by investigators and journal editors of the deleterious effects pathogens have on research results. Overall, the data point to the conclusion that progress toward elimination of pathogens is not keeping pace with the current sophistication in biomedical science. REFERENCES Allen, A. M., and T. Nomura, eds. 1986. Manual of Microbiologic Monitoring of Laboratory Ani- mals. NIH Pub. No. 86-2498 (USDHHS, NIH, DRR). Government Printing Office, Washing- ton, D.C. Baker, D. G. 1998. Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clin. Microbiol. Rev. 11:231-266. 

J. RUSSELL LINDSEY 43 Bhatt, P. N., R. O. Jacoby, H. C. Morse III, and A. E. New, eds. 1986. Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research. Academic Press; Orlando, Fl. 844 pp. Casebolt, D. B., J. R. Lindsey, and G. H. Cassell. 1988. Prevalence rates of infectious agents among commercial breeding populations of rats and mice. Lab. Anim. Sci. 38:327-329. Jacoby, R. O., and J. R. Lindsey. 1997. Health care for research animals is essential and affordable. FASEB J. 11:609-614. Jacoby, R. O., and J. R. Lindsey. 1998. Risks of infection among laboratory rats and mice at major biomedical research institutions. ILAR J. 39:266-271. NRC [National Research Council]. 1991. Infectious Diseases of Mice and Rats. A report of the Committee on Infectious Diseases of Mice and Rats. National Academy Press, Washington, D.C. 397 pp. NRC [National Research Council]. 1998. Biomedical Models and Resources: Current Needs and Future Opportunities. A report of the Committee on New and Emerging Models in Biomedical and Behavioral Research. National Academy Press, Washington, D.C. 45 pp. Webster’s Ninth New Collegiate Dictionary. 1989. Merriam-Webster Inc., Springfield, MA. P. 771. Weisbroth, S. H. 1996. Post-indigenous disease: Changing concepts of disease in laboratory rodents. Lab. Anim. 25:25-33.