Description of RIKEN BDR SPF Barrier Facilities
We, RIKEN BDR, started to use our Animal Facility in September 2003. The facility is designed for housing mice and rats and is managed separately by room or area. The health status of the animals is determined by health monitoring tests in sentinel animals, which are conducted periodically to confirm that there are no disease outbreaks in the facility.
All live mice are obtained from approved vendors (Oriental Yeast Co., Ltd., CLEA Japan Inc., Japan SLC, Inc., and SHIMIZU Laboratory Supplies Co., Ltd.). Occasionally we obtain mice from other sources (i.e., universities and/or private industries), and these animals are always obtained through IVF and embryo transfer techniques to prevent contamination.
Access to the facility is controlled by ID cards; therefore, only authorized users are able to enter animal rooms. Personal protective equipment (PPE; sterile gown, disposable hair bonnets, gloves, facemasks, and shoe covers) must be worn to enter the facility. All items going into the facility are sterilized or decontaminated (wiped clean with an appropriate disinfectant). Animals are maintained in autoclaved cages placed in ventilated racks and are provided with irradiated food and filtrated water. The stations, which are working space, are used for cage changing.
RIKEN BDR Animal Facility’s Policy Concerning Mouse Microbiological Standards
The risks of contamination from the transfer of animals should be avoided by microbial monitoring on the distribution side and by quarantine (or isolation) on the reception side; however, there are limits to risk avoidance on both sides. With regard to the distribution side, differences in quality standards are a problem. Research facilities do not have standardized lists of microbes that are regularly checked for or lists of microbes that are considered dangerous. Testing procedures and quarantine methods also vary, and no international standards have been established to control the specificity or sensitivity of these checks. As a result, quarantine must be carried out to determine whether or not the quality of the animals received conforms with one’s own quality standards. Aside from quarantine, it may be necessary to eliminate infected animals by IVF techniques. To reduce complex procedures and possible confusion, we have adopted the following policies regarding mouse transfer and health monitoring procedures.
RIKEN BDR Animal Facilities Policy
To increase the scientific reliability of experiments conducted at RIKEN BDR, and to ensure ethical conduct of animal experiments, we will establish a microbiological profile (quality standard) for breeding mice.
Microbial monitoring must be carried out in order to: (1) ensure that quality is maintained according to established standards, (2) prevent the outbreak and spread of infectious diseases in the research facilities, and (3) avoid unnecessary problems when transferring mice.
To ensure that the results of the animal monitoring are objective, we will ask a third party to perform the testing.
In principle, transferring mice as frozen embryos is preferred.
If requested by the reception side, we will provide guidance on techniques for thawing frozen embryos.
When transferring live mice, we will disclose the results of all microbiological examinations. By taking this information into account, we will make every effort to promote the safe and smooth transfer of transgenic mice.
In regard to the responsibility of preventing contamination by infectious agents when transferring animals between scientific colleagues, we consider that both parties involved in the animal transfer share this responsibility equally.
Background
In the following attachment from the ICLAS Monitoring Center (attached to the Central Institute for Experimental Animals), microbes and parasites that are known to infect mice are classified into categories A-E based on their virulence.
A: Zoonotic pathogens from mice that can cause illness in humans
B: Pathogens that are lethal to mice
C: Pathogens that are non-lethal to mice but have the potential to alter physical/immunological functions (with apparent or non-apparent symptoms).
D: Opportunistic pathogens that can cause illness in mice under certain conditions (irradiated mice, immunosuppressed mice, etc.)
E: Non-pathogenic parasites that serve as an indicator of facility contamination.
Since the above list of organisms can be quite extensive, ICLAS has prepared the “Core set items” list from the above categories. This list has been narrowed down according to prevalence in Europe, America, and nationally, then divided into groups based on detection method: serology, cultivation, microscopy. We are conducting periodic monitoring of organisms on this list.
RIKEN BDR Mouse Microbiological Profile
Due to their virulence towards people or mice, microbes belonging to the abovementioned category A or B pose a threat to both research development and animal welfare, and every effort must be made to eliminate them. Some of the microbes from categories C, D, and E may give us useful information to identify the microbiological status of animals at the time of transfer, and should also be included in the monitoring. Additionally, we must check for other microbes and parasites (from categories C, D, or E) if requested by the facilities receiving our mice. Furthermore, we cannot ignore organisms that might adversely affect immunocompromised host mice, especially if the contamination prevalence of such organisms is high. By taking the above into consideration, we have decided to monitor selected microbes and parasites belonging to categories C, D, or E, in addition to those in A and B. Using the above mentioned notes as a foundation, guidelines concerning the CDB mouse microbial profile are outlined below.
Core Battery
We have chosen a total of seven microbes from categories A and B of the ICLAS list of microbes. From category A: Lymphocytic choriomeningitis virus, Salmonella spp. From category B: Ectromelia virus, Mouse hepatitis virus, Sendai virus, Mycoplasma pulmonis, and Citrobacter rodentium. These microbes are included in the Core Battery of RIKEN BDR Mouse Pathogens because they pose a significant threat to both the facility personnel and the mouse colony. To maintain our colony and to avoid risk of contaminating other facilities, we will work to ensure that these organisms do not contaminate our facilities. Thus, all examinations for the Core Battery of organisms must always produce a negative result (no contaminating organisms present). This level of quality is the standard at RIKEN BDR mouse facilities.
Supplemental Battery
Aside from microorganisms included in category A or B, we will also monitor microbes and parasites that are included in the above mentioned ICLAS monitoring set, according to contamination prevalence. We have chosen Clostridium piliforme, Corynebacterium kutscheri, Pasteurella pneumotropica, Intestinal protozoa from category C, as well as Helminth and External parasites in category E. Although these are not included in the Core Battery of RIKEN BDR Mouse Pathogens, they are listed in the Supplemental Battery as organisms to be monitored for useful information. The results of monitoring for the Supplemental Battery of microorganisms will be disclosed to those facilities receiving our organisms.
Spot Check
In addition to regular monitoring of the above mentioned microbes, spot checks will be performed according to necessity or demand. We will conduct spot checks for seven murine viruses (from EDIM virus to reovirus type 3 in the attached Microbe Checklist), and four bacteria (from CAR bacillus to Staphylococcus aureus), as well as dermatophytes Pneumocystis carinii and Aspiculuris tetraptera. The full list of microbes that we will conduct spot checks for are listed under “Spot Check” in the Microbe Checklist.
Examination enforcement and disclosure of the examination results.
Please refer to the Microbe Checklist for further details on the following categories of microorganisms:
Core Battery
We will request periodical examinations conducted by a third party at least 4 times a year. The results of this monitoring must be negative. If it becomes clear that the colony has been contaminated with these organisms, we must eliminate the colony and reestablish it from frozen embryos or by in vitro fertilization.
Supplemental Battery
We will request periodical examinations conducted by a third party at least 2 times a year. The results of the Supplemental Battery tests are not as serious as those of the Core Battery. If contamination by these organisms has been identified, we will breed the infected animals separately and start a new, uncontaminated colony by in vitro fertilization, or isolate the contaminated colony and work hard to replace it. We will permit mice from these colonies to be transferred; however, receiving facilities will be informed with a clear warning that these organisms may be present.
Spot Check
No regular monitoring. We will do spot checks for this class of organisms according to demand, or according to the particular immunological conditions of the mice. The results of the spot checks will be used for information purposes, as they do not warrant extreme measures in response. However, if contamination has been identified, we will consider “cleaning” the infected colony via in vitro fertilization.
Summary
In the past, when contamination by a particular organism was discovered through monitoring, the policy was to destroy the colony immediately. Due to such policies, scientists often hesitated to check for mild pathogens. We have recognized that there are some organisms which must be checked for even though their detection does not necessitate extreme measures. Thus, we have established a more comprehensive list of organisms with three distinct levels of security:
| Core Battery | organisms dangerous enough to warrant the immediate elimination of the colony. |
| Supplemental Battery | controllable organisms that require isolation, but not immediate elimination of infected colonies. |
| Spot Check | possibly harmful organisms not checked by the ICLAS’s “Core set items” list, which will be checked for, when necessary. |
Finally, to prevent the spread of infection, animals will be transferred as frozen embryos, in principle. If the receiving facility wishes, we will consider the transfer of live animals. In all cases, we will disclose the results of the microbial monitoring and spot checks.
Microbe Check List
Core Battery
Dangerous organisms which must be eliminated (To monitor periodically)
| Lymphocytic choriomengitis virus（リンパ球性脈絡髄膜炎ウイルス） | Category A |
| Salmonella spp.（サルモネラ） | Category A |
| Ectromelia virus (エクロトメリアウイルス） | Category B |
| Mouse hepatitis virus (マウス肝炎ウイルス） | Category B |
| Sendai virus (センダイウイルス） | Category B |
| Mycoplasma pulmonis (肺マイコプラズマ） | Category B |
| Citrobacter rodentium (腸粘膜肥厚症菌） | Category B |
Supplemental Battery
Organisms to be monitored as useful information (To monitor periodically)
| Clostridium piriforme (ティザー菌) | Category C |
| Corynebacterium kutscheri (ネズミコリネ菌) | Category C |
| Pasteurella pneumotropica (肺パスツレラ） | Category C |
| Intestinal protozoa (消化管内原虫) | Category C |
| Helminth (蠕虫類) | Category E |
| External parasites (外部寄生虫) | Category E |
Spot Check
To check as needed/requested
EDIM (Rota) virus, Minute virus of mice/Mouse parvovirus, Mouse adenovirus, Mouse cytomegalovirus, Mouse encephalomyelitis virus GDVII, Pneumonia virus of mice, Reovirus Type 3, Cilia-associated respiratory (CAR) bacillus, Helicobacter hepaticus/bilis, Pseudomonas aeruginosa (緑膿菌), Staphylococcus aureus, Dermatophytes(皮膚糸状菌), Pneumocystis carinii (カリニ原虫), Aspiculuris tetraptera (ネズミ大腸蟯虫)