animal research a risky business. However, steady progress in improving the health and genetic quality of laboratory mice and rats since the 1960s has reduced this risk and enhanced the value of animal experimentation in virtually every field of biology and medicine. Great progress occurred not only in the detection, elimination, and prevention of common pathogens, but also in the genetic manipulation of the mouse and rat through sophisticated breeding schemes. These advances provided access to novel mutants such as widely used models of immune dysfunction. Thus, as the century draws to a close, laboratory mice and rats have become vanguards of animal-based research. They are small enough (and big enough), tame enough, fertile enough, cheap enough, healthy enough, and genetically uniform enough to meet critical standards for mammalian modeling.

These attributes were less obvious for a while, not very long ago. Advances in molecular biology and biotechnology, especially during the 1970s and 1980s, were viewed by some as a harbinger of reduced reliance on vertebrate animal experimentation. In vitro or invertebrate alternatives offered opportunities for cheaper and faster answers to some scientific questions. In fact, many institutions experienced a decline in animal research during those decades. This trend occurred despite the fact that genetics, neoplasia, immunology, metabolism, and a host of other areas remained well suited to exploration in vertebrate models. But doubts about the relevance of vertebrate animal research are now moot, because, ironically, many of the tools and concepts that suggested imminent tempering of animal-based research became stimulants for an explosive growth in animal use. Molecular and developmental biologists put the mouse genome ''in motion'' and changed the face and potential of animal-based research dramatically and permanently. The impact of the genetically altered mouse, which is still a scientific infant, and its cousin, the genetically altered rat, which is by comparison a scientific fetus, is reflected in their anointment, with "digestible" hyberbole, as the "E. colis" of the 21st century.

The advent of genetically altered rodents, however promising scientifically, also is associated with biological, technological, logistical, and financial challenges that are emerging at an astounding rate. The challenges for assuring biologic integrity in genetically altered animals are dealing with intervening infections in diverse environments using diverse assessment standards and diverse terminology. Most of these challenges stem from the development, characterization, production, distribution, housing, husbandry, and health care associated with novel animals. And they raise a fundamental question: With so many genetically new animals being developed and used in so many places, by so many people, so quickly, how can their biological integrity be defined and ensured? The following remarks attempt to highlight briefly some of the issues flowing from this question with the expectation that others at this meeting will address them in greater depth.

My definition of "biological integrity" is incomplete, but, for the moment, consider the term to mean "the stability of intrinsic and extrinsic factors that

define the structural and functional characteristics of an animal." Therefore, the benchmarks for defining a laboratory rodent in the era of genetic engineering must include at least the establishment, standardization, and monitoring of factors such as genotype, phenotype, microbial status, and environmental quality. Criteria such as reproductive capacity and other health-related factors such as susceptibility to infection should also be considered.

These concepts also imply that biological integrity can be perturbed by intrinsic or extrinsic interference, which may be overt or subtle. This threat is especially relevant considering the diversity of settings in which genetically engineered rodents are being made. Variability can be caused by genetic drift; the influence of genetic background on the penetrance of a phenotypic trait; opportunistic infection that may be pathogenic, disruptive to normal responses, or conducive to erroneous phenotyping; environmental stresses such as noise, vibration, and threatening odors; and many other factors. Variability also can be abetted by diverse or ill-defined terminology. For example, and as noted elsewhere in these proceedings (Lindsey, 1999), the term "specific pathogen free" has lost value because of the lack of precision with which it often is employed and perceived. Additionally, the increased use of animals inherently increases risks to biological integrity from dense housing and increasing exchanges of animals and animal products among laboratories, nationally and internationally.

Because worldwide reliance on laboratory rodents will increase for the foreseeable future, internationally standardized criteria and definitions should be developed as benchmarks for the biological integrity of laboratory rodents. A number of questions should be answered in formulating a transnational strategy to achieve this goal, a few of which are cited here. What are the criteria and definitions that should be used to measure biological integrity? Which assessments should be performed and how often? Who should perform the assessments? How should assessment results be reported and accessed? What sources are available to support research and development of new or improved assessment methods? Who should be responsible for funding assessment programs and how can the funds be leveraged for maximum benefit to biomedical research and the health of laboratory animals?

The time is ripe for international cooperation and action on these important issues. Meetings such as the US/Japan conference in session today can and should play a central role in getting planning under way.

Animal resources directors of major American universities. 1998 annual workshop. Cincinnati, Ohio.

Lindsey, J. R. 1999. Current status of pathogen status in mice and rats. Pp. 39–43 in Proceedings of the US/Japan Meetings, October 23, 1998. National Academy Press, Washington, DC.

NCRR [National Center for Research Resources], National Institutes of Health. 1997. The national survey of laboratory animal use, facilities and resource. USPHS, Washington, DC.

Toshio Itoh

Deputy Director, ICLAS Monitoring Center

Central Institute for Experimental Animals

Kawasaki, Japan

The International Council for Laboratory Animal Science (ICLAS), the only international organization related to laboratory animal science, designated our institute as an ICLAS Monitoring Center in 1979. It is currently the only such center.

In Japan, most of the mice and rats used in experiments are specific pathogen-free (SPF) animals supplied by breeders. In animal experimentation facilities, barrier systems for maintenance of SPF animals are also widespread. However, there are no uniform standards in academic associations concerning quality testing systems for SPF animals. Several organizations have prepared recommendations for a quality testing system including test items, test frequency, and sample size. Those organizations are the ICLAS Monitoring Center, the Association of Laboratory Animal Facilities of National Universities, and the Japanese Society of Laboratory Animals. Breeders and users have established their own quality testing systems using these recommendations as a reference. However, because the staff of the ICLAS Monitoring Center participated in the preparation of these associations' recommendations, the quality testing systems of organizations that actively undertake quality control are basically the same as that of the ICLAS Monitoring Center.

The organization of the ICLAS Monitoring Center is as follows. An Advisory Board has been established in the Center to hear outside opinions. The members of this Board are the following six organizations: the Association of

Laboratory Animal Facilities of National Universities, Japanese Association for Experimental Animal Technologists, Japanese Association for Laboratory Animal Science, Japanese Association of Experimental Animals, Japanese Society of Laboratory Animals, and Japan Pharmaceutical Manufacturers Association. They include the main groups of laboratory animal breeders and users. The Center consists of three divisions: genetics, microbiology, and embryo bank. The operating funds are obtained as income from monitoring and cryopreservation services ordered by animal facilities of commercial breeders, pharmaceutical companies, universities and research institutes, as well as government support. Last year the Center received support from the Ministry of Education, Science, Sports and Culture of Japan.

In 1997, the activities of the microbiology division of the Center were as follows. Microbiological monitoring was performed on about 18,000 samples from about 1,700 animal production facilities and animal experimentation facilities. The Center also produced and supplied antigens, antisera, and antibody testing kits as reference substances indispensable in microbiological monitoring. About 1,000 vials of antigens and antisera and about 3,000 testing kits were distributed. The Center held two workshops with academic societies, gave lectures in universities and institutions, and jointly held training courses with various organizations in an effort to promote monitoring.

The antibody testing kit uses enzyme-linked immunosorbent assay (ELISA) produced and supplied by the Center and can be used for testing four microbes: Sendai virus, mouse hepatitis virus, Mycoplasma pulmonis , and Tyzzer's organism (Clostridium piliforme). These items were selected because of their importance as pathogens and their prevalence in Japan. The Center, which is the only organization in Japan that has its own antigens and antisera and system for microbiological testing, performs testing on a third party basis.

Overseas, the East Asian countries are still in a rather weak position, but their economies have expanded remarkably in recent years. In these countries, substantial progress has been made in science and technology. Assistance provided by the Center in the field of laboratory animals in these countries includes receiving trainees, on-site education, guidance, and a supply of reference substances. Since 1979, the Microbiology Division has accepted 16 trainees from Asian countries. By means of these activities in Japan and abroad, the ICLAS Monitoring Center has become a center for the quality control of laboratory animals, not only in Japan but also in East Asia.

There do not appear to be any major differences in sampling size and frequency of monitoring between Japan and the West, but there are slight differences in the criteria used for selection of test items. In the United States and

especially Europe, all microorganisms that might affect experimental results are tested. However, we also take into consideration the pathogenicity of the microorganism for the animal, the possibility of transmission and disease in humans, and the opportunity for infection based on the contamination map. Because laboratory animals kept in barrier facilities are monitored, we do not think it is necessary to include parasites that require an intermediate host or microorganisms for which spontaneous infections have not been confirmed and the possibility of infectious disease has been found only in infection experiments. Monitoring also requires economic considerations. I do not think it is necessary to monitor all items at all times in all animal facilities; nor is it necessary for the tests to be the same for breeders and users. For example, breeders should supply as much information (test results) as possible on the animals of interest to the user since it is not clear for what experiments the animals will be used. However, researchers as users need only test results for microorganisms that might cause damage to the experiment. The quality control systems are basically different for animal experiments using immunodeficient animals and for those with little burden placed on the animals.

Our concept for selection of test items in the microbiological monitoring system of the Center is based on categorization of test items as described in the Manual of Microbiologic Monitoring of Laboratory Animals (US Public Health Service/NIH 1994). For the reasons I mentioned above, the microorganisms we test for are classified into five categories. Category A consists of zoonotic and human pathogens carried by animals; category B consists of pathogens fatal to animals; category C consists of pathogens that are not fatal but can cause disease in animals and affect their physiological functions; category D consists of opportunistic pathogens; and category E consists of indicators of the microbiological status of an animal or colony. The microorganisms to be tested should be selected based on the degree of microbiological control in each animal facility. For example, in SPF animal production facilities, as many items as possible including all categories are selected; in animal facilities requiring strict microbiological control, such as those performing experiments using immunodeficient animals or experiments placing a heavy burden on the animals, categories A, B, C, and D are selected; and in facilities undertaking experiments with little burden on the animals, categories A and B are sufficient. The test items performed in our Center on mice and rats are listed by category in Table 1. In the selection of test items in individual animal facilities, consideration should be given to the possibility of in-house testing, outsourcing of the testing, current status of microbiological contamination, and which experiments are being performed.

I present here our recent test results and compare them with those of the

United States. The facilities that asked the Center to perform the tests included breeders and animal experimentation facilities. Our results reflect the microbiological quality of laboratory animals in Japan.

There are three large and several small SPF animal breeders in Japan. The microbiological quality of animals in these SPF animal breeders has basically been maintained in good condition free from test items of categories A, B, C, and

E. However, infections do occur in SPF animal breeders. Contamination by M. pulmonis or Pasteurella pneumotropica has recently been seen in several SPF breeders.

Our results in mouse and rat experimental facilities can be seen in Tables 2 and 3. Among our categories A, B, and C, category A: zoonosis was never found, but contamination by pathogens in categories B and C have been observed in

mouse experimental facilities. The highest contamination rates are seen for mouse hepatitis virus and P. pneumotropica. The number of positive items and their positivities are lower in pharmaceutical companies than in universities and institutes. In mouse experimental facilities, infections have been decreasing with the spread of the barrier system, and such infections have basically disappeared in pharmaceutical companies. However, in universities and research institutes where introduction of the barrier system has been delayed, there are still sporadic infections.

In rat experimental facilities, among our categories A, B, and C, category A was never found; but contamination by pathogens in categories B and C has been observed. Main pathogens detected in rats were M. pulmonis, Clostridium piliforme, cilia-associated respiratory (CAR) bacillus, and sialodacryoadenitis virus. The positive items and their positivities showed the same trends as those in mouse experimental facilities.

The microbiological quality of mice and rats between US and Japanese experimental facilities is shown in Figures 1 and 2. US results were quoted from "Health Care for Research Animals Is Essential and Affordable" in FASEB Jour-

Figure 1

Comparison of microbiological quality of mice between the United States  and Japan (% positive of agents in animal facilities).