by Ron Dattoli (Wyeth, Cambridge, MA) with assistance from Adam Warner (BIOQUELL, Inc.)
The following technical article was given as a poster presentation at the 2004 National Meeting of the American Association for Laboratory Animal Science and describes events that occurred between January 2002 and October 2004.
Maintaining a viral-free environment is probably one of the more difficult tasks in any laboratory rodent facility. The most effective form of protection from a viral outbreak is awareness. Educating all staff in the facility regarding how viruses can enter the facility and the impact viral infections have on animal studies is vital. This, along with ensuring that all personnel adhere to practices, policies, and procedures, is essential to achieving and maintaining a viral-free environment. Traffic flow within the facility and personal protective wear also play a major role in maintaining a viral-free rodent facility.
Mouse parvovirus (MPV) is one of the more common viruses to infect laboratory rodent facilities. Although it is common practice to screen cells lines or their products for murine viruses prior to in vivo administration using mouse antibody production (MAP) or polymerase chain reaction (PCR) tests and to purchase only viral-free rodents, viruses still find their way into animal facilities. Once a virus is detected in a facility, determining its mode of entry can be extremely difficult. The source could be wild rodents that got into the facility, contamination of laboratory rodents while they were in transit, employees who maintain rodents at home, visitors from other animal facilities, or equipment brought from laboratories outside the animal facility or other institutions.
After a virus has entered a facility, keeping it contained with eventual eradication is achievable if appropriate procedures are in place. This article describes the actions taken to decontaminate mouse rooms and procedure rooms in response to different outbreaks of MPV and focuses on comparisons of para formaldehyde and hydrogen peroxide (H2O2) for decontamination of rooms and equipment.
Materials and Methods
During the past several years, one or more sentinel mice periodically tested positive for MPV using serology to detect antibodies. Our procedures for dealing with MPV positive sentinels and decontaminating functional areas have changed over the years. In the late 1990s, when sentinel mice became positive for MPV at the old Cambridge facility or newer Andover facility, the procedure was to “quarantine” the room, test representative mice, eliminate any mice that tested positive, remove the “quarantine” and resume normal operations. MPV continued to appear in both facilities two or three times a year. In other words, this approach did not eliminate MPV.
At the end of 2000, the recurrent MPV outbreaks prompted a closer look at traffic flow, decontamination procedures, and chemicals and detergents used in the Andover facility. Our procedure changed to strict quarantine of the mouse room while the initial positive serological test was confirmed using additional serological tests for antibody or PCR to detect virus in tissues. When confirmation of MPV was obtained, depopulation and decontamination of the room was scheduled following completion of any studies in the room.
Decontamination consisted of the following: all soiled caging was emptied into trash bags inside the biological safety cabinet (BSC) in the room. Bags were sealed and sprayed with chlorine dioxide. Once the breakdown was complete, the entire room was jet sprayed with quaternary ammonium disinfectant at a concentration of one ounce per gallon. With all equipment remaining in the animal room, the room was fogged with chlorine dioxide, mixed one part base, one part activator, and three parts water. Upon completion of fogging, all equipment that could be autoclaved was autoclaved and then sent through the cage wash. All disposable items were autoclaved and discarded. The room was then brought back on line. However, MPV reoccurred in the Andover facility in spite of these measures.
It appeared a combination of quaternary ammonium disinfectant followed by fogging with chlorine dioxide did not completely eradicate MPV. It was decided to use a peracetic acid/hydrogen peroxide cold sterilant for routine sanitization of hoods and to replace fogging oxide with hand-spraying of peracetic acid/hydrogen peroxide cold sterilant. But there were still recurrences in the Andover facility and some possible contributing factors were found.
Late in 2002, it was decided to use para formaldehyde gas to decontaminate “MPV areas” in the Andover facility after hand-sanitation with quaternary ammonium disinfectant and a peracetic acid/hydrogen peroxide cold sterilant. A contractor was hired to perform the para formaldehyde decontamination, which seemed to be effective because the Andover facility has been free of MPV for the past two years. However, it was expensive and, since the gas is toxic, our EH&S personnel had to work with the contractor to seal the area before para formaldehyde application and monitor the area before people reentered.
During the two-year period following the opening of a new facility in Cambridge, MPV was detected on several occasions. Most episodes resolved after strict quarantine, depopulation, hand-sanitation with quaternary ammonium disinfectant followed by spraying peracetic acid/hydrogen peroxide cold sterilant and para formaldehyde decontamination. However, a second episode of MPV in the same room prompted consideration of additional measures.
We thought the virus was surviving on investigators’ equipment, some of which had been moved down from the Andover facility. A method of decontamination that could safely permeate the sensitive (and expensive) equipment (Figure 1) without causing damage was needed. Hydrogen peroxide (H2O2) vapor decontamination has been shown to be effective in the eradication of viruses in animal facilities. 1-3
At this point, HPV equipment was purchased from a selected vendor and technical support staff were sent to assist in the initial decontamination procedure. This was particularly important initially to determine the correct concentration of H2O2 for the specific areas requiring decontamination.
The space was prepared by first purging all disposable equipment and items but leaving all racks, turbo units, computers and electronic equipment in place. Doors to cabinets and drawers were left open to maximize the permeation of H2O2. Our internal HVAC staff closed all necessary dampers to ensure personnel and animal safey in other areas of the facility. EH&S was also involved and provided information as to the allowable limits of H2O2 concentration before re-entering the suite.
Biological indicators (G. stearothermophilis discs) were placed in numerous locations within the space, particularly in areas that were considered to be “difficult” to reach. The HPV generator was set to run over a period of several hours (Figure 2). This includes the gassing, dwelling and aerating phases. Once the concentration of H2O2 was determined to have reached safe levels (1 ppm), entry was made into the space. The G. stearothermophilis indicators were retrieved, incubated in tripticase soy broth and observed for growth once daily for seven days. Caging, racks, turbos and other non-electronic equipment were removed and sanitized before being returned to service.
G. stearothermophilis indicators exposed to H2O2 vapor during facility decontamination were negative (no growth) on culture and unexposed controls were positive (growth) on culture. This indicated that the decontamination process was effective. Since H2O2 leaves no residual, there was no need to wipe down the electronic equipment, countertops or other surfaces.
Another outbreak of MPV occurred in the same area approximately three months after the initial H2O2 decontamination. Evaluation suggested some research equipment, especially that stored in cabinets or boxes, might not have been adequately exposed. During the next H2O2 decontamination, the electronic equipment and other research equipment was placed in a biosafety cabinet that was fitted with a special panel across the sash (Figure 3). All equipment or supplies were raised off of cabinet shelves and counter tops.
Identifying the source and point of entry of viruses into a rodent facility is a difficult task. A regular, scheduled health monitoring program combined with regular, scheduled cleaning and sanitization are extremely important in maintaining a viral-free environment. Periodic decontamination of “at risk” areas with a chemical sterilant should be considered during development of a sanitation program.
Hydrogen peroxide (H2O2) vapor is an excellent choice for microbial decontamination of environmental surfaces in an unoccupied room. The effectiveness of decontamination by H2O2 vapor can be evaluated easily through the use of biological indicators placed at key locations. In comparison to formaldehyde gas, hydrogen peroxide vapor leaves no residue, acts more rapidly, and is easier to evacuate from the treated space. Cost analysis reflects substantial savings over time.
Ed Acosta, Mercedes Collazo, Dominica Lopes and Richard Zapata for all their hard work during depopulation, sanitation and decontamination following a MPV episode. Tony Battaglino, Tracy Lewis, and Dr. Terrie Cunliffe- Beamer for assistance. Adam Warner & Mike Herd of BIOQUELL,Inc.
About the Authors
Ron Dattoli has been a supervisor at Wyeth in Cambridge, MA for the last seven years and a member of AALAS since 1996. He also is a active member of NEBALAS and became an AALAS Certified LAT in 2002.
Adam T. Warner is the Eastern United States Technical Manager for BIOQUELL, Inc. He specializes in hydrogen peroxide bio-decontamination of rooms, isolators, safety cabinets, and various pieces of laboratory equipment and has presented training seminars for AALAS, PDA, ABSA, and ISPE.