The two topics of my discussion are examples. The first topic is an example of contamination of a human tumor line passaged in immunodeficient mice, which illustrates the effects of Helicobacter hepaticus infection on animal experiments. The second topic is not actually related to H. hepaticus infection, but I use this organism to illustrate a method for establishing test items when a new infection appears in laboratory animals.
H. HEPATICUS CONTAMINATION OF HUMAN TUMOR TISSUES PASSAGED IN IMMUNODIFICIENT MICE
One aspect of cancer research at the Central Institute for Experimental Animals involves the collection of tumor tissues from patients and the subcutaneous inoculation into immunodeficient animals such as nude mice or mice with severe combined immunodeficiency (SCID) mice. In this process of passaging human tumor tissues using immunodeficient mice, focal necrosis appears very often in the liver of the cancer-bearing mice after a certain period. I describe below the characteristics of the infected mice, the process of the infection, and the countermeasures against it.
Although bacteriologic and histopathologic tests were performed using mice with liver lesions, and serum antibody tests were performed using sentinel mice to detect known pathogens, the cause of the lesions was not clear at the start of testing. After H. hepaticus was recognized as a new mouse pathogen and after a test method was established in our laboratory, it was confirmed that the cause of this abnormality was infection with this organism. According to results of tests on the cancer-bearing mice, the lesions were the same as those reported previously in immunodeficient mice, that is, focal liver necrosis and proliferative colitis. Curved bacilli were detected in the bile canaliculi surrounded by necrosis and in the crypts of the large intestine.
The specific DNA sequence of this organism was detected by polymerase chain reaction (PCR) in livers (20/21), cecal contents (21/21), and transplanted tumors (19/20) in these cancer-bearing mice. It was assumed that H. hepaticus bacteremia occurred in the infected immunodeficient mice similarly to the occurrence of Helicobacter bacteremia in AIDS patients. We first determined when H. hepaticus was introduced into the colony. PCR for H. hepaticus in cryopreserved tumors strongly suggested that the contamination occurred in 1990, as shown in Table 1. Based on the time of the tumor contamination and facility records, we suspect that this organism was brought into the facility by genetically engineered mice introduced from overseas.
We then investigated eliminating this organism from the contaminated tumor tissues. We passaged the tumor tissues collected from these infected animals directly into noncontaminated SCID mice under sterile conditions; however, after several weeks, we found that the organs and tumors of the mice were H. hepaticus DNA positive. In subsequent investigations, after cryopreserved samples of H. hepaticus DNA-positive tumor tissue were thawed and transplanted into SCID mice, we found that these mice were H. hepaticus free, and this organism had also been eliminated from the transplanted tumor tissues. From these results, it was assumed that the SCID mice had bacteremia due to H. hepaticus infection and the transplanted tissue was also contaminated. However, the bacterial count in each cryopreserved sample was low, the bacteria in the tissue were killed by the freeze-thawing process, and no infectious bacteria remained even though the tissue was contaminated. After that, we confirmed that the process caused a decrease in the viable number of H. hepaticus to 10−1 or 10−2.
We found that in transplantation studies of tumors and organs using immunodeficient mice, H. hepaticus also caused persistent bacteremia in the same way that mouse hepatitis virus, Sendai virus, or Mycoplasma pulmonis infection causes viremia or bacteremia. As a result, the tumor and organ samples were contaminated. H. hepaticus should be considered one of the organisms that requires precautions in animal experimentation facilities. I would like to emphasize the importance of quarantine for biomaterials from immunodeficient animals.
ESTABLISHMENT OF TEST AGENTS
In the establishment of test agents for microbiologic monitoring, the issue is whether to establish individual test agents for each species (such as H. hepaticus or bilis) or to establish the tests for the genus as a whole (such as Helicobacter spp.). According to the FELASA recommendations, tests for Pasteurella spp. are specified for mice and rats, but tests for individual species (such as Pasteuella pneumotropica and multocida) are not specified. Several species of Helicobacter have been detected in mice, and some of these have not been confirmed as pathogenic. However, it appears that testing for Helicobacter spp. will be adopted in Europe and the United States. Therefore, I would like to present my thoughts concerning the selection of test agents with respect to international harmonization of quality control of laboratory animals.
I propose that organisms subject to testing in the microbiologic monitoring of laboratory animals be assigned a significance and be divided into the five main categories described in the Manual of Microbiologic Monitoring of Laboratory Animals, published as a result of prior US-Japan Meetings (USPHS/NIH 1994). These categories are A: zoonotic and human pathogens carried by animals; B: pathogens fatal to animals; C: pathogens not fatal but that can cause diseases in animals and affect their physiologic functions; D: opportunistic pathogens for animals; and E: indicators of the microbiologic status of an animal or colony.
At the ICLAS Monitoring Center, we believe that test agents should be selected for definite reasons. We therefore have established the following list for obtaining necessary information: whether the organism has been confirmed to be pathogenic, and effects on the experimental results are clear; convenience of testing (whether ordinary test methods are established and kits are available); prevalence or usefulness as an indicator of microbiologic control. An overly long list of test agents will result in an increase in costs over the whole range from production to the use of laboratory animals. Continuing qualitative improvement of laboratory animals cannot be expected with such an increase in costs. With respect to animal experimentation facilities in particular, the problem is how many users actually have facilities that can utilize such sophisticated animals.
In the harmonization of quality tests, it is clear that selection of test agents is the most important factor. Therefore, test agents adopted for the genus should not be increased aimlessly, and caution is required. Overtesting is of benefit only to testing facilities and some large-scale producers; it does not necessarily improve the quality of laboratory animals and animal experimentation, which is our objective. In the selection of test agents, I believe that additional consultation is necessary among researchers performing animal experiments, animal breeders, and testing facilities.
USPHS/NIH [US Public Health Service/National Institutes of Health] Manual of Microbiologic Monitoring of Laboratory Animals. 2nd ed. Washington, DC: GPO; 1994. NIH Publication No. 94-2498.
National Academies Press (US), Washington (DC)
Itoh T. Emerging Diseases in Mice and Rats. In: National Research Council (US) International Committee of the Institute for Laboratory Animal Research. Microbial Status and Genetic Evaluation of Mice and Rats: Proceedings of the 1999 US/Japan Conference. Washington (DC): National Academies Press (US); 2000.