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Microbial Status and Genetic Evaluation of Mice and Rats: PROCEEDINGS OF THE 1999 US/JAPAN CONFERENCE Standardization of Rodent Health Surveillance: Regulation Versus Competition William Shek Charles River Laboratories Wilmington, MA INTRODUCTION Standardization may result from regulation or “recommendations” (in quotes because the recommendations are often followed as if they were regulations). In Europe, an attempt is being made to standardize rodent health monitoring through recommendations promulgated by the Federation of European Laboratory Animal Science Associations (FELASA) (Rehbinder and others 1996). These recommendations, which include lists of infectious agents and standard report formats by species, were developed to simplify the evaluation of rodent health status, irrespective of source. However, some have misunderstood the purpose of the recommendations by concluding that rodents infected with any of the microorganisms on the FELASA reference list are not suitable for use in research. Such misunderstanding underscores the danger of presenting simple lists of infectious agents to people who do not have a general understanding of laboratory animal microbiology. The relevance of lists of microorganisms, such as those published by FELASA, is further compromised when they are not regularly updated to include the latest findings in laboratory animal microbiology, a very active field of research. For example, helicobacters are not included in the FELASA list. Even recently prepared lists will be more applicable to certain situations than to others. In addition, they inevitably reflect the bias and limited knowledge of the cadre who create them. Another approach is to standardize to the diagnostic reagents and methods of a reference laboratory. Dependable reagents from a trusted and well-known source can be especially valuable when the relevant resources and technical
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Microbial Status and Genetic Evaluation of Mice and Rats: PROCEEDINGS OF THE 1999 US/JAPAN CONFERENCE expertise are not available, especially if the reagents are provided in a simple-to-use kit format. Standardized reagents and assay methods might also lead to fewer discrepancies in the results from different laboratories, but such strict standardization is not without significant risks. No assay is completely sensitive and specific. Often the deficiencies in an assay become known only when comparative testing is performed in multiple laboratories that use different reagents and test methods. In the 1980s, the first evidence for the existence of additional rodent parvovirus serotypes came from laboratories employing indirect immunofluorescence assays (IFA) to detect rodent parvovirus antibodies, instead of the more commonly used hemagglutination-inhibition tests (HAI) and enzyme-linked immunosorbent assays (ELISA). The better sensitivity of the IFA for detecting cross-reacting parvovirus antibodies is related to the nature of the IFA antigen, which is composed of infected cells that contain large amounts of the highly conserved and, hence, cross-reactive nonstructural (NS) parvoviral proteins. Certainly in this case, had all laboratories been using recommended assays and reagents, the discovery of the new parvovirus serotypes (i.e., mouse parvovirus [MPV] and rat parvovirus [RPV]) would have been delayed substantially. In the United States, there is minimal governmental regulation of laboratory animal testing, although the USDA does license veterinary diagnostic test kits, including those developed to test rodents. Despite the dearth of regulatory oversight, there have been continual and substantial improvements in the health of laboratory rodents and the breadth and quality of rodent diagnostic services. The contention made here is that these improvements and de facto standardization have come about, and will continue to occur, because of competition among laboratory animal suppliers, diagnostic laboratories, and researchers. Furthermore, regulations and recommendations implemented by governmental agencies and professional organizations might impede the incorporation of recent research advances into current laboratory animal husbandry and health surveillance practices. ANIMAL HEALTH Husbandry As noted, the quality of specific-pathogen-free (SPF) laboratory rodents supplied by major animal breeders has improved steadily and dramatically in the United States with minimal government regulation. For example, most vendors have switched in recent years from raising severe combined immunodeficiency mice and athymic nude mice in barrier rooms to raising them in isolators in which a restricted microflora, free of opportunistic pathogens, can be sustained. This costly change was not made in response to a mandate from a government agency or professional organization. Rather, it was implemented to meet the demands of researchers for healthier animals and to stay competitive.
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Microbial Status and Genetic Evaluation of Mice and Rats: PROCEEDINGS OF THE 1999 US/JAPAN CONFERENCE As investigators have become more cognizant of the expanding body of scientific studies showing the complications to research that are caused by adventitious infections, biosecurity has improved at research facilities as well. Many institutions have adopted the use of microisolation cages, and it appears that the prevalence of adventitious infections has dramatically decreased at these institutions. Surveillance Without prescriptions from governmental agencies, commercial and non-commercial laboratory animal vendors are screening their colonies for largely the same infectious agents, including most exogenous viruses, primary and opportunistic bacteria, and ecto- and endoparasites. With regard to sample size and frequency, it appears that serology for viral antibodies is performed on a monthly to quarterly basis, whereas other types of health monitoring, bacteriology, and parasitology are performed on a quarterly basis. Many factors such as husbandry practices and the historical incidence of contamination, however, affect whether sample size and the frequency of testing are adequate. Statistical models that take into consideration the effect of current husbandry practices (such as the use of microisolators and sentinels kept on pooled, soiled bedding) on sampling are just not available. Therefore, governmental and professional organizations have no sound basis from which to mandate or recommend sample size or sampling frequency. Results Reporting The reporting of results has also become standard among laboratory rodent vendors. Generally, vendors report health surveillance results by room and species. The panel of microorganisms included in the reports is largely consistent from vendor to vendor. Reports are generally divided into three sections: serology, bacteriology, and parasitology. Most suppliers have two report categories, specific pathogen free (SPF) and additional agents. SPF reports include those agents that must be excluded from a colony because of documented health and research effects. The additional agents report may include opportunists and other agents of interest to researchers. Results are presented as the number positive over number tested. Reports typically show cumulative data, perhaps over a 12-month period; the most recent test results may also be presented. Sometimes animal strains in the room are listed in the report. Plan of Action When an adventitious infection is found, a vendor's action is largely determined by customer requirements and the microbial status of competitor colonies.
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Microbial Status and Genetic Evaluation of Mice and Rats: PROCEEDINGS OF THE 1999 US/JAPAN CONFERENCE A vendor might compile certain lists of agents for which immediate or planned depopulation is mandated because of pathogenicity, research effects, or customer preferences. We recently detected seroconversion to Theiler's mouse encephalomyelitis virus (TMEV) in a rat colony. Essentially nothing is known about the cause of TMEV seroconversion in rats. We suspect a related picornavirus is responsible for seroconversion, but our attempts to isolate or detect virus by animal inoculation or polymerase chain reaction (PCR), respectively, have been unsuccessful. Despite this and a dearth of information in the laboratory animal science literature, once informed of our serologic findings, customers chose not to receive rats from the affected colony. Hence, customers essentially determined that we had to eliminate the colony. Of course, a vendor's actions also depend on financial considerations. If exclusion of a particular microorganism becomes important to the research community but all of a supplier's colonies are infected with that agent, the supplier cannot be expected to immediately depopulate all affected colonies. However, we have observed in many instances that competition from suppliers with colonies free from infection with a particular pathogen will cause others to replace their infected colonies. HEALTH SURVEILLANCE Methodologies All or most diagnostic laboratories have by now converted to using for serology the sensitive, nonradioisotopic, solid-phase immunoassays developed in the 1980s, such as the ELISA and IFA. Among the molecular, or DNA, methodologies that have recently been applied to infectious disease diagnosis, the PCR has become the most popular. In rodent diagnostics, the University of Missouri Research Animal Diagnostic and Investigative Laboratory has led the way and has the largest panel of PCR assays for rodent infectious agents. Most other diagnostic laboratories, including ours, are following suit by developing PCR for viruses and other microorganisms. From the comparative results of rodent diagnostic quality control program, we know that there can be great disparities among the test results reported by different laboratories. Comparative serology results recently reported by the European Laboratory Animal Health Monitoring Club, however, showed substantial agreement among laboratories, albeit for a limited number of viruses and other microorganisms (Dix and Needham 1996). Microorganisms Emerging rodent pathogens are continually being discovered by laboratory animal microbiologists. Without regulation or recommendations, how do rodent diagnostic laboratories respond to these findings? In the 1980s, the important
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Microbial Status and Genetic Evaluation of Mice and Rats: PROCEEDINGS OF THE 1999 US/JAPAN CONFERENCE newly discovered agents were MPV and RPV, originally known as the “orphan” parvoviruses (Jacoby and others 1996). The pathogenesis of MPV in particular has been elucidated at Yale's Section of Comparative Medicine, which it is worth noting received substantial support in this effort from a commercial vendor. At this time, most if not all laboratories offer serology for MPV and RPV antibodies, primarily by ELISA with recombinant antigens and by IFA. More recently, research done at various institutions has shown that infections with certain species of Helicobacter may cause disease, especially in immunodeficient mice. Without any regulations or recommendations requiting them to do so, most rodent diagnostic laboratories in the United States (and in Europe and Japan as well) quickly developed and began offering Helicobacter PCR assays. SUMMARY In the United States (and I would argue in Europe and Japan as well), the main motivation for standardization among the suppliers of rodents and diagnostic services has been competition and not government regulation or the recommendations of professional organizations. There is strong competition among microbiologists, laboratory animal suppliers, and diagnostic laboratories to discover and publish on important etiologic agents, to provide the highest quality SPF rodents, and to offer the most complete and accurate testing services, respectively. Laboratory animal suppliers have had to conform to the de facto standards that are set by their competitors and the demands of the research community. Consequently, there is little variation among suppliers of the excluded infectious agents that define rodents as SPF. To keep up with their competitors, diagnostic laboratories have had to quickly adopt the latest assay methodologies and to add tests for emerging pathogens. Concordance of the results reported by different laboratories for assays to diagnose common infections is good. Nevertheless, a quality assurance program to assess the accuracy of laboratory results, such as the one discussed in this meeting by Dr. Riley, is sorely needed. REFERENCES Dix, J., and J.R. Needham. 1996. Assessing the impact needs reliable results: The Laboratory Animal Health Monitoring Club. Scand. J. Lab. Anim. Sci. 23:171-176. Jacoby, R.O., L. Ball-Goodrich, D.G. Besselsen, M.D. McKisic, L.K. Riley, and A.L. Smith. 1996. Rodent parvovirus infections. Lab. Anim. Sci. 46:370-380. Rehbinder, C., P. Baneux, D. Forbes, H. Van Herck, W. Nicklas, Z. Rugaya, and G. Winkler. 1996. FELASA recommendations for the health monitoring of mouse, rat, hamster, gerbil, guinea pig and rabbit experimental units. Lab. Anim. 30:193-208.
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