4th National Symposium on Biosafety

Animal Biosafety Levels 1-4: An Overview

Jonathan Y. Richmond, PhD
Director, Office of Health and Safety
Centers for Disease Control and Prevention
1600 Clifton Road, F0-5
Atlanta, GA 30333
404 639-2453

This presentation will be a very rapid run through of the current biosafety recommendations for persons working with vertebrate animals.

The slides used in this presentation will be available for persons who are involved with training. Since training is a real integral part of almost all successful programs today, we have reproduced them in this Proceedings as CDC's gift to you (see Appendix).

Yesterday Dr. Martin Favero said that the most frightening words in the English language are "I'm here from the government and I'm here to help you". I just wanted to make sure that you realize we really are here to help you. I will be joined in a few moments by Dr. Bobby Brown, a CDC veterinarian, who will pick up where I leave off. I will begin with the introduction to Animal Biosafety Levels 1 and 2. Bobby will talk about ABL-3 and then give you a visual perspective of some of the ABL-4 issues.

Vertebrate Animal Biosafety Levels are designated as ABSL-1, 2, 3 and 4. These are based on a combination of recommendations that have grown out of experience when we talk about the practices and procedures which are so crucial daily.

We talk about safety equipment or the primary barriers; the facilities themselves - how they are designed and run - are the secondary barriers. This would be applicable for all vertebrate animals for experiments where we are using infectious agents, specifically those that will cause infections in humans. The ABSLs provide for increasing levels of personnel and environmental protection, and they are to be considered as minimal standards for safe work with infected laboratory animals. You take the information that's in the CDC/NIH guidelines (Biosafety in Microbiological and Biomedical Laboratories) (BMBL) as Step 1, and then you adopt it to your own facilities and make it user-friendly.

We need to use procedures to minimize both the splatters and the aerosols such as using a cotton pledget to cover the withdrawal of a needle from a vial. Animal Biosafety Level 1 also defines some special practices such as decontaminating all your work surfaces after use or after a spill of viable materials. We must be cognizant of appropriate decontamination of equipment. For example, enrichment equipment that's used with non-human primates may need to be washed in soap and water and decontaminated. It is essential to be very careful about handling animal waste because animal care personnel may develop medical problems following prolonged exposures to proteins in urine.

Cages can be washed manually or in a cage washer. The final rinse should be at a 180º F, in conjunction with the Guide. Doors to animal rooms should open inward and have a self-closing mechanism on them. Doors should be kept closed when experimental animals are present. We don't like to see the little kick stands on the bottom of doors to keep them propped opened. Obviously, if you have to move cages in and out, you need to hold doors open, but truly the doors should be kept closed. You need to have an insect and rodent control program in place. Many of the newer control programs involve use of a lot fewer chemicals for insect and rodent control, preferable to just continuing with a spray program.

We want to prevent persons who are at increased risk of acquiring infection from entering our animal rooms. We are dealing increasingly with populations of employees who may have lifestyles or medical conditions that put them at increased risk of exposure to certain things. People who are immunocompromised or who may be on certain drug therapies that induce immunosuppression are at increased risk. However, this also creates an opportunity for the supervisor of the facility to really understand the employees working there.

We also want to be careful about allowing animal room access for persons whom infection might be unusually hazardous. For example, we don't believe that it is a good idea to have children come in to look at the new ferret babies. It's much better to visit a petting farm for appropriate animal exposure. There is interest in having our children understand our personal work environments, but we must also minimize their risks.

We have no recommendations for primary barriers of Biosafety Level 1 because they are not needed. Animal Biosafety Level 1 is for work with agents that are not particularly hazardous for healthy adult humans; therefore, we don't need special containment.

For Animal Biosafety Level 2, all of the standard practices indicated for ABL1 remain the same. A key point to keep in mind is that what is said for Biosafety Level 1 applies at 2, and then the sum of those apply at 3 and so on. There are some special practices at Biosafety Level 2:

For example, if immunization, a baseline serum, or wearing special clothing is required in the animal facility, that all needs to be posted. It's not just the animal care people and the veterinarians who have access to animal facilities. It might also be the maintenance personnel or, heaven forbid, an investigator who actually wants to see their animals.

Serological surveillance programs are of interest, and a later session will speak very specifically about this. If you have a baseline serum storage program, you need to have a formal written program in place. With the emerging litigious society that we're in, things like informed consent and other legal documents need to be on file. Things that didn't apply 25 years ago do today, so be aware and be prepared.

You may want to set up specific testing protocols for sera at very defined intervals. This may well be for a pre-exposure or post-exposure incident or for somebody who has just been exposed to some agent. You can take a baseline serum at that point and 28 days later look for rising titer or whatever your protocol says you should do. The key to this is maintaining written records. You need to place those written records in the official medical folder of the employee, and you need to be conveying this information to the supervisor. At CDC we have developed an automatic immunization and medical record tracking system which is kind of nice. If you visit the CDC, we'll be able to talk a bit more about the system we call AIMS. Finally, the employee obviously needs to be kept in the loop recording any medical records and the availability of results of testing.

If you are going to conduct a [medical] surveillance program, you'll need to define things such as:

These are interesting issues that you need to work on with your legal people. Obviously, an appropriate immunizations component could include anything from Hepatitis A to B, and/or it may be very specific. For example, if you have a project on rabies, you would want to vaccinate employees. We would also include TB skin testing in the same medical surveillance program.

You need to maintain the written medical evaluation, any surveillance data that you collect on testing, and any treatment data resulting from any associated occupational injury or illness. Immediately report spills and accidents! Nelson Garnett described a problem with perception as to whether an event had been an accident or an intentional injury.

At CDC, we believe that they all are incidents that need to be reported and evaluated. This is really helpful for the person who may wind up at some point needing to file a worker's compensation claim for insurance reasons, etc.

Training has to be provided, at least annually, to personnel who are involved with animal care and use issues. This training should cover:

If you have an SOP and for some reason you're going to change it, it is crucial to gather personnel together to explain the changes that will occur. You should document the fact that you have training and that specific individuals have been trained. I know that for those of you who are in the training business, it is a difficult thing to make the second and third years of training interesting, exciting and titillating.

You also have to prepare or adopt a biosafety manual. The CDC/NIH guide, (the BMBL), is a minimum guide, and we hope that you will take the information that's there and customize it to work for you. Clearly, I don't know what your Building 47 is, what your Room 3 is, or where the incinerator is, etc. For persons who would like to receive a copy of this book, or if you would like to receive a Word Perfect disk version, we will mail it to you. The reason I offer this is that it's always easier to plagiarize, and I'd prefer that you plagiarize correctly rather than misstate what we have taken so much time to write in the book.

We certainly need to be posting signs for persons who are going to enter the laboratory or animal holding space. Other information that might be included on the sign:

People know they must wear appropriate gowns, gloves, respirators, and other kinds of personal protective equipment no matter where they go inside the facility, but you must make sure that they don't wear these items outside of the animal facility. The clothing is worn to protect the person, and in some cases it's worn to protect their street clothes. However, protective clothing or equipment should not be worn to the cafeteria, the library, etc. Protective clothing is worn in the animal room and is removed before leaving the facility.

A wide variety of personal protective devices are on the market. You're aware of the incredible array of gloves to protect against a wide variety of njuries. I simply caution you to make sure that the gloves that you are using are appropriate for the agents or the chemicals that may be in use. Other kinds of personal protective equipment would include goggles, eye protection, face shield, possibly a respirator if required and so forth. These protective equipment needs must be worked out in your safety meetings as you plan and initiate the research experiments.

At Biosafety Level 2, we want to be particularly careful about needles and syringes. We want to make sure that we are substituting plastic for glass ware. The other day somebody asked me about the use of Pasteur pipettes, and I suggested that while there are some cardboard sleeves in which you could dispose of those pipettes, there are also plastic Pasteur pipettes with self-contained bulbs that you can use to minimize the problems of sticking yourself or dropping and breaking glass equipment.

We need to have some mechanism for wrapping up the materials that we move from the animal facility back to the laboratory room. There are a number of devices on the market. This carrying case has a rubber gasket, and it is a commercially available device. You can put in a rack to hold your vacutainers or other tubes, load, close it up, wipe it off with a suitable disinfectant, and transfer it then to the laboratory where you would then process material in a biological safety cabinet. Please decontaminate all contaminated equipment before reuse or, in particular, before removal from the animal facility. Autoclaving is perhaps the most common way to decontaminate something, but we recognize that there are a wide variety of other ways, including use of some chemicals.

Cages get autoclaved before cleaning at ABSL-2. Animals not involved in the work being performed must be kept out of the facility. That's probably not a very big issue, but you'd be surprised how many people like to bring pet animals into laboratories, and that becomes an issue.

Safety equipment, the primary barriers that we recommend, are biological safety cabinets (BSC). We have a new publication that was just released on proper selection and use of biological safety cabinets. More and more you see BSCs popping up in animal procedure rooms. They are most appropriate in some cases for transferring animals and in some cases for dissecting necropsies.

The secondary facilities are basically the same as we would see at Level 1, except we begin to pay some attention to the way air is moved in this environment. We want to maintain directional inward air flow. We want to make sure that the animal room itself is negative to the corridor so that air moves from the corridor (the area of least contamination) to the area of potentially higher contamination. This also does wonders for odor control in the corridors. The air should be exhausted to the outside without recirculation to other rooms. In some of our facilities at CDC, we have cut slots in the doors, and have hung a little piece of surveyor's colored tape. As long as that tape flutters inward, the animal care people know that they are maintaining that inward directional air flow. If the air flow direction falls static or if its reverses itself, then they know to call engineering to rectify the problem.

You want to make sure that the facilities are designed and constructed to facilitate cleaning and housekeeping. This becomes a big issue and has to be appropriate for the species involved. Finally, in many of our animal rooms we have drains with traps in the floor. If you ave drains, you have to have a trap in there; you also want to make sure that you fill that trap with at least water. You can also put a good disinfectant down there, If you aren't hosing down your rooms or if you're basically leaving them static once you've put your disinfectant in, you may want to pour a cup of mineral oil on top to minimize evaporation. Mostly what we're concerned about is growth of bacteria in the sewer, but if a trap dries out, then you get gas in the room and that really is obnoxious.

This very rapid run through of Animal Biosafety Levels 1 and 2, paints the picture for what we do when we have to deal with agents that are beyond "just agents" such as hepatitis. We're moving into areas like working with tuberculosis in animals. What about work with more hazardous microorganism at Animal Biosafety Levels 3 and 4? I now present Dr. Bobby Brown, head of our Animal Resources Program at CDC, who will continue this dialog.

References:

CDC/NIH, Biosafety in Microbiological and Biomedical Laboratories, 3rd edition,

CDC/NIH, Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets, HHS Pub, US Government Printing Office. Washington, September, 1995.

Office of Health and Safety, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Mail Stop F05 Atlanta, Georgia 30333, USA Last Modified: 1/2/97
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Animal Biosafety Levels 1-4: An Overview

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