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Quality Testing System for SPF Animals in Japan and Problems in the Management of Such Systems Toshio Itoh Deputy Director, ICLAS Monitoring Center Central Institute for Experimental Animals Kawasaki, Japan ROLE OF THE ICLAS MONITORING CENTER IN THE QUALITY CONTROL SYSTEM OF LABORATORY ANIMALS 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 test- ing systems for SPF animals. Several organizations have prepared recommen- dations for a quality testing system including test items, test frequency, and sample size. Those organizations are the ICLAS Monitoring Center, the Associ- ation 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 associationsâ recommendations, the quality testing systems of organiza- tions 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 Advi- sory Board has been established in the Center to hear outside opinions. The members of this Board are the following six organizations: the Association of 15
16 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE 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 oper- ating 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 facili- ties. 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 Tyzzerâs organ- ism (Clostridium piliforme). These items were selected because of their impor- tance 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 pro- vided by the Center in the field of laboratory animals in these countries includes receiving trainees, on-site education, guidance, and a supply of reference sub- stances. 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. MICROBIOLOGICAL MONITORING SYSTEM OF THE ICLAS MONITORING CENTER There do not appear to be any major differences in sampling size and fre- quency of monitoring between Japan and the West, but there are slight differ- ences in the criteria used for selection of test items. In the United States and
TOSHIO ITOH 17 especially Europe, all microorganisms that might affect experimental results are tested. However, we also take into consideration the pathogenicity of the micro- organism 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 microorgan- isms 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 experi- ments 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 oppor- tunistic 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 ani- mals, 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 con- tamination, and which experiments are being performed. MICROBIOLOGICAL CONTAMINATION OF LABORATORY ANIMALS IN JAPAN I present here our recent test results and compare them with those of the
18 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE TABLE 1 Selected Microbes for Monitoring Categorya Microbes Mice Rats (no. of organisms) 24 microbes 24 microbes A(4) Dermatophytes X X Hantavirus X Lymphocytic choriomeningitis (LCM) virus X Salmonella spp. X X B(5) E. coli 9115a, c:k(B) X Ectromelia virus X Mouse hepatitis virus X Mycoplasma pulmonis X X Sendai virus X X C(18) Clostridium piliforme X X Bordetella bronchiseptica X Cilia-associated respiratory (CAR) bacillus X X Corynebacterium kutscheri X X Giardia muris X X H-1 virus X Kilham rat virus X Minute virus of mice X X Mouse encephalomyelitis virus X X Mouse adenovirus X X Pasteurella pneumotropica X X Pneumonia virus of mice X X Reovirus type 3 X X Sialodacryoadenitis virus X Spironucleus muris X X Streptococcus pneumoniae X Heliobactor hepaticus X Lactic dehydrogenase virus X D(2) Pseudomonas aeruginosa X X Staphylococcus aureus X X E(1) Syphacia spp. X X aCategory A: Pathogens that might infect humans. Category B: Pathogens fatal to animals. Category C: Pathogens not fatal, but can cause diseases in animals and affect their physiological functions. Category D: Opportunistic pathogens. Category E: Indicators of the microbiological status of an animal or colony. United States. The facilities that asked the Center to perform the tests included breeders and animal experimentation facilities. Our results reflect the microbio- logical 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
TOSHIO ITOH 19 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 TABLE 2 Microbiological Monitoring in Mouse Experimental Facilities (1992-1996) Facilities Pharmaceutical Universities/ Categorya Items companiesb % Institutesb % B Mouse hepatitis virus 52/599 8.7 221/910 23.2 Sendai virus 1/599 0.2 8/910 0.9 Mycoplasma pulmonis 2/599 0.3 20/910 2.2 C Pneumonia virus of mice 3/599 0.5 2/910 0.2 Mouse encephalomyelitis virus 0/599 2/910 0.2 Mouse adenovirus 0/599 1/910 0.1 Clostridium piliforme 0/599 1/910 0.1 Corynebacterium kutscheri 0/599 1/910 0.1 Pasteurella pneumotropica 17/288 5.9 55/352 15.6 Giardia muris 0/215 1/222 0.5 Spironucleus muris 0/215 2/222 0.9 D Pseudomonas aeruginosa 59/288 24.2 50/352 20.2 Staphylococcus aureus 80/155 51.1 26/70 37.1 E Syphacia obvelata 12/215 5.6 11/222 5.0 Facilities A Dermatophytes 0/209 Hantavirus 0/51 Salmonella spp. 0/640 Lymphocytic choriomeningitis (LCM) virus 0/79 B Ectromelia virus 0/1509 E. coli 0115a, c:k(B) 0/640 C Minute virus of mice 0/62 Cilia-associated respiratory (CAR) bacillus 0/64 Helicobacter hepaticus 4/12 aCategory A: Pathogens that might infect humans. Category B: Pathogens fatal to animals. Category C: Pathogens not fatal, but can cause diseases in animals and affect their physiological functions. Category D: Opportunistic pathogens. Category E: Indicators of the microbiological status of an animal or colony. bNo. of positive facilities/no. of tested facilities
20 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE TABLE 3 Microbiological Monitoring in Rat Experimental Facilities (1992-1996) Facilities Pharmaceutical Universities/ Categorya Items companiesb % Institutesb % B Sendai virus 3/694 0.3 14/315 4.4 Mycoplasma pulmonis 1/694 0.1 29/315 9.2 C Pneumonia virus of mice 2/694 0.3 1/315 0.3 Sialodacryoadenitis virus 0/694 20/315 6.3 Cilia-associated respiratory (CAR) bacillus 0/19 1/15 6.7 Clostridium piliforme 38/694 5.5 28/315 8.9 Corynebacterium kutscheri 2/694 0.3 0/315 Pasteurella pneumotropica 2/337 0.6 2/315 0.6 Giardia muris 0/204 1/60 0.5 Spironucleus muris 1/204 0.6 3/60 1.7 D Pseudomonas aeruginosa 62/337 18.4 15/315 4.8 Staphylococcus aureus 103/145 66.9 7/21 33.8 E Syphacia muris 12/204 5.9 21/49 45.7 Facilities A Dermatophytes 0/186 Hantavirus 0/279 Salmonella spp. 0/652 Lymphocytic choriomeningitis (LCM) virus 0/26 C H-1 virus 0/37 Kilham rat virus 0/38 Mouse adenovirus 0/1009 Mouse encephalomyelitis virus 0/1009 Bordetella bronchiseptica 0/337 Streptococcus pneumoniae 0/652 aCategory A: Pathogens that might infect humans. Category B: Pathogens fatal to animals. Category C: Pathogens not fatal, but can cause diseases in animals and affect their physiological functions. Category D: Opportunistic pathogens. Category E: Indicators of the microbiological status of an animal or colony. bNo. of positive facilities/no. of tested facilities
TOSHIO ITOH 21 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 âHealth Care for Research Animals Is Essential and Affordableâ in FASEB Jour- The U.S. Bacteria Japan CAR bacillus Mycoplasma Helicobacter Parasites Pinworms LCM Adeno Viruses Sendai PVM TMEV MHV 0 10 20 30 40 50 60 70 Percent FIGURE 1 Comparison of microbiological quality of mice between the United States and Japan (% positive of agents in animal facilities).
22 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE Bacteria The U.S. Japan CAR bacillus Mycoplasma Pinworms Parasites MAV Viruses TMEV Sendai PVM Corona 0 10 20 30 40 50 60 Percent FIGURE 2 Comparison of microbiological quality of rats between the United States and Japan (% positive of agents in animal facilities). nal by Drs. Jacoby and Lindsey (1997), who are present today. These results were compared for tests performed in both countries. The trends were the same for mice and rats. There were fewer positive items in Japan than in the United States, and when the tests were positive, the positive rates were lower in Japan. Japan is a small country where it is comparatively easy to reach a consensus. Once such a consensus is reached, there is a tendency to persevere in the direction decided. This national characteristic may be the reason that quality control of laboratory animals in Japan has been more successful than in the United States. CONCLUSION Internationalization of laboratory animals has made remarkable advances as seen with genetically engineered animals, and mice and rats can be shipped all over the world by air. From the standpoint of animal control, there are now more opportunities for infected animals or materials obtained from infected animals to enter facilities. In Japan currently, Pneumocystis carinii pneumonia and viral hepatitis in immunodeficient mice and in immunological function knockout mice introduced from the United States have become a major problem in microbiologi- cal control in animal facilities. In the United States, contamination of sera with
TOSHIO ITOH 23 ectromelia virus has presented a problem. Animal quality control, especially microbiological monitoring, is becoming more important for maintenance of laboratory animals and assuring reproducibility of experimental results. REFERENCES Jacoby, R., and J. R. Lindsey. 1997. Health care for research animals is essential and affordable. FASEB J. 11:609-614. US Public Health Service/NIH. 1994. Manual of Microbiologic Monitoring of Laboratory Animals. 2nd edition. (NIH Publication No. 94-2498). GPO, Washington, D.C.