（A）BSL-4実験室での安全上のご注意と操作手順：3.空中生物学

The overall goal of this protocol is to provide a general understanding of animal biosafety Level IV cabinet laboratory aerosol procedures that support the safety of laboratory researchers. Visualizing this protocol helps show the complexities and safety components to health administrators who are contemplating the design of similar buildings and to outside collaborators involved in studies of high consequence pathogens. The main advantage of this technique using negative-pressure Class III biosafety cabinets for aerosol experiments is that the laboratory staff can safely conduct aerobiology research without positive-pressure suits.

As the lead aerobiologist, I will be demonstrating the procedure along with Kyle Bohannon, the aerosol engineer from our laboratory. Test the functionality of the rapid transfer port, or RTP, equipment involved in aerobiology procedures including transport cart doors and the RTP connecting the transport cart through the wall to the Class III biosafety cabinet, or BSC. Lock the RTP into place.

Monitor and record any physical manipulations, administrations, or routine procedures on charts for each nonhuman primate, or NHP. If additional anesthesia is required, ensure that all removed caps, used needles, sharps, and syringes are discarded in a sharps container. Do not recap any needles after use.

Transport anesthetized NHPs in clear containers that are secured by a latch on the lid of the transport box. Load the transport containers onto a mobile cart to allow fully-suited researchers to move freely using breathing airlines into the air pressure-resistant doors. When an aerosol challenge is completed, place the NHP inside the transport cart and return the NHP to the home cage.

Concurrently with animal preparation performed by comparative medicine staff, test the functionality of the decontaminated biosafety cabinet. Verify that negative-pressure in the Class III BSC is maintained within the specified range. Inspect the Class III BSC for any potential leaks or cracks.

Inspect the synthetic rubber gloves and o-rings attached to the decontaminated Class III BSC for weak spots, tears, rips, or dry rot. At this stage, the Class III biosafety cabinet is not contaminated. Replace the damaged Class III BSC synthetic rubber gloves and/or o-rings.

Inspect the dunk tank and verify that it is filled with disinfectant to the marked level, either three inches above the immersion baffle or to a marked line inside the dunk tank. Verify the concentration of disinfectant in the dunk tank is a minimum of 3, 500 microsiemens, or 3.5 millisiemens, using a conductivity meter. Ensure the Class III BSC autoclave is functional to receive contaminated waste and equipment when the experiment is completed or to pass for one-time only overlooked equipment needed for the experiment into the Class III BSC from the autoclave portal.

Autoclave only the equipment known to sustain the rigors of the process. Test the functionality of other aerobiology equipment. For example, the electronic connections and outlets.

Place gloved hands within the synthetic rubber gloves attached to the Class III BSC. Assemble the NHP head-only exposure chamber, the conditioning tube, and the aerosol management platform. Attach an aerosol generator to the conditioning tube and the aerodynamic particle sizer to the NHP head-only exposure chamber.

Retract gloved hands from the attached synthetic rubber gloves. Discard personal gloves as biohazardous waste each time gloved hands are removed from the attached synthetic rubber gloves. Next, launch the aerosol protocol software on the laptop computer.

Enter the appropriate NHP head-only exposure chamber, aerosol generator, and biosampler flow rate, as well administrative information into the software menus. Receive the already prepared pathogen through the rapid transfer port and fill the aerosol generator with the pathogen. The Class III biosafety cabinet is now contaminated.

Through the aerosol software, turn the aerosol generator on. The pathogen from the aerosol generator will fill the exposure chamber. After turning off the aerosol generator, empty the challenge material and discard it into a biohazardous trash bag located inside of the Class III biosafety cabinet.

Next, fill the biosampler with collection media and attach the filled biosampler to the NHP head-only exposure chamber. Attach the appropriate vacuum line to the biosampler. Check that the depth of anesthesia of the NHP is adequate as demonstrated by unresponsiveness to external stimuli, muscular tone, and stable respiratory and heart rates.

Pass the anesthetized NHP through the RTP into the Class III BSC. Place the NHP in the supine position onto the exposure ramp. Verify that the NHPs vital signs are stable visually and with a portable subject monitor.

Calibrate for respiratory inductive plethysmography, or RIP, prior to the experiment using the manufacturer's protocol. Place the respiratory inductive plethysmography bands around the abdomen and chest of the NHP. Check that these bands are properly fitted and the electronic connections are snapped tightly.

Next, place the Numatech calibration mask over the snout of the NHP. Then check that a seal is made around the NHPs snout. Calibrate the RIP bands with a Numatech calibration mask using computer software.

Upon completion, remove the calibration mask. Record the respiratory parameters and export the respiratory data for each animal through a compatible program. Gently pass the NHP's head through the rubber dental dam attached to the head portal of the NHP head-only exposure chamber.

Ensure that the rubber dental dam creates a seal around the NHP's neck during the aerosol exposure. Enter the target aerosol accumulated volume and necessary equipment identifiers pertinent to each exposure run into the software and initiate the aerosol procedure. Once the NHP breathes in the target accumulated volume, the system shuts down.

Unhook the RIP bands and lay them on the bed next to the NHP. Wipe with NHP with gauze soaked in one to two percent chlorohexidine solution, exercising caution when wiping around the NHP's eyes. Dab the head of the NHP to remove excess solution.

Pass the NHP back through the RTP to the researchers inside the ABSL-4 aerobiology suit laboratory. Empty the aerosol generator and any of the remaining challenge material into the biohazardous trash bag containing trash, disposable equipment, and 3/4-full sharps containers. Empty the collection media from the aerosol biosampler into the appropriately labeled collection tubes.

Place the collection tubes on wet ice in a bucket for the appropriate bio-assay to determine an estimated inhaled dose. And pass the sample collection tubes and bucket through the RTP. The Class III biosafety cabinet is a hermetically sealed stainless steel cabinet under negative-pressure within an animal biosafety Level IV cabinet laboratory.

Materials are introduced into the biosafety cabinet by staff working in the animal biosafety Level IV cabinet laboratory through an under-cabinet mounted stainless steel tank, or dunk tank, containing a five percent dual quaternary ammonium disinfectant solution. Because the BSC is built into the wall separating the cabinet laboratory from an ABSL-4 suit laboratory, materials, animals, and viral pathogens can also be moved into the BSC from the suit laboratory side, using a transport cart and an RTP. Once the transfer cart doors are opened to the ABSL-4 suit laboratory, the Class III biosafety cabinet is considered contaminated.

Items within the BSC can be manipulated from the outside by staff wearing various types of synthetic rubber gloves. These items, excluding infectious samples, are removed from the BSC after sterilization through a double-door autoclave or disinfection via the dunk tank. By verifying that the Class III biosafety cabinet and bio-aerosol equipment are functioning properly, a safe and properly operational environment is maintained that is integral to the safety of the staff.

Through strict adherence to these procedures and practices, no laboratory acquired infections have been recorded during bio-aerosol research at the Integrated Research Facility, Frederick. Once mastered, this technique can be done in a timely manner to allow for multiple nonhuman primate exposures in one day. While attempting this procedure, remember to maintain an adequate depth of anesthesia.

Without steady state sedation, the plethysmography acquisition becomes much more challenging. Following this procedure, other aerobiology experiments involving smaller species such as rabbits and mice, can be performed. This technique paves the way for aerobiology researchers to control variables that affect dosimetry, such as temperature, relative humidity, and biosampler efficiency, to deliver a precise inhaled dose.

After watching this video, you should have a good understanding of the biosafety precautions used during aerobiological experiments. Precautions such as multiple personal glove changes should decrease potential laboratory acquired infections.