Name: safety precautions and operating procedures in an (a)bsl-4 laboratory: 3. aerobiology
Uploaded: 2016-10-03T11:00:00
Description: Watch this Scientific Journal Video about Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology at JoVE.com

The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services (DHHS) or of the institutions and companies affiliated with the authors. This work was funded in part through Battelle Memorial Institute's prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I. J.K.B., K.J., M.R.H., D.P., L.B., and J.W. performed this work as employees of Battelle Memorial Institute. Subcontractors to Battelle Memorial Institute who performed this work are: J.H.K., an employee of Tunnell Government Services, Inc.; and M.G.L., an employee of Lovelace Respiratory Research Institute.

This protocol adheres to the following animal care guidelines. Animals were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All experimental procedures were approved by the National Institute of Allergy and Infectious Diseases, Division of Clinical Research, Animal Care and Use Committee and were in compliance with the Animal Welfare Act regulations, Public Health Service policy, and the Guide for the Care and Use of Laboratory Animals ...

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	<li>Alibek, K., Handelman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The chilling true story of the largest covert biological weapons program in the world-told from inside by the man who ran it. , (1999).</li><li>Roy, C. J., Pitt, L. M., Swearingen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+disease+aerobiology:+aerosol+challenge+methods.">Infectious disease aerobiology: aerosol challenge methods.</a> Biodefense: research methodology and animal models. , 61-76 (2006).</li><li>Lackemeyer, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABSL-4+aerobiology+biosafety+and+technology+at+the+NIH/NIAID+integrated+research+facility+at+Fort+Detrick.">ABSL-4 aerobiology biosafety and technology at the NIH/NIAID integrated research facility at Fort Detrick.</a> Viruses. 6 (1), 137-150 (2014).</li><li>Bohannon, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+characterization+of+large-particle+aerosols+using+a+center+flow+tangential+aerosol+generator+with+a+non-human-primate,+head-only+aerosol+chamber.">Generation and characterization of large-particle aerosols using a center flow tangential aerosol generator with a non-human-primate, head-only aerosol chamber.</a> Inhal Toxicol. , (2015).</li><li>Chosewood, L. C., Wilson, D. E., eds, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological and Biomedical Laboratories. , (2009).</li><li>Janosko, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+Precautions+and+Operating+Procedures+in+an+(A)BSL4+Laboratory:+1.+Biosafety+level+4+suit+laboratory+suite+entry+and+exit+procedures.">Safety Precautions and Operating Procedures in an (A)BSL4 Laboratory: 1. Biosafety level 4 suit laboratory suite entry and exit procedures.</a> J Vis Exp. , (2015).</li><li>Mazur, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+Precautions+and+Operating+Procedures+in+an+(A)BSL4+Laboratory:+2.+General+Practices.">Safety Precautions and Operating Procedures in an (A)BSL4 Laboratory: 2. General Practices.</a> J Vis Exp. , (2015).</li><li>Mortola, J. P., Frappell, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+barometric+method+for+measurements+of+ventilation,+and+its+use+in+small+animals.">On the barometric method for measurements of ventilation, and its use in small animals.</a> Can J Physiol Pharmacol. 76 (10-11), 937-944 (1998).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+respiratory+inductive+plethysmography+module+supporting+multiple+sensors+for+wearable+systems.">Development of a respiratory inductive plethysmography module supporting multiple sensors for wearable systems.</a> Sensors (Basel). 12 (10), 13167-13184 (2012).</li><li>Ingram-Ross, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiorespiratory+safety+evaluation+in+non-human+primates.">Cardiorespiratory safety evaluation in non-human primates.</a> J Pharmacol Toxicol Meth. 66 (2), 114-124 (2012).</li><li>Besch, T. K., Ruble, D. L., Gibbs, P. H., Pitt, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steady-state+minute+volume+determination+by+body-only+plethysmography+in+juvenile+rhesus+monkeys.">Steady-state minute volume determination by body-only plethysmography in juvenile rhesus monkeys.</a> Lab Anim Sci. 46 (5), 539-544 (1996).</li></ol>

We outline the aerobiology procedures used at the IRF-Frederick for working with highly hazardous (Risk Group 4) pathogens. One purpose of visualizing the bioaerosol procedures is to emphasize the safety of staff when using a Class III BSC during experimentation with such pathogens to avoid laboratory-acquired infections. Class III BSCs maintain an inward directional airflow that exhausts into double HEPA filters to ensure that pathogens are contained within the laboratory (Figure 1).
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Transmission of viruses generally occurs by direct or physical contact, but many important viral diseases (e.g., measles, chickenpox, influenza) are caused by pathogens that are transmitted by aerosol or respiratory droplets. Such pathogens have the potential to cause a pandemic with consequences ranging from widespread mild disease associated with loss of work (e.g., common cold) to rarer serious disease with high lethality (e.g., smallpox). High-consequence pathogens that spread naturally by aerosol or by intentional aerosol release (biological weapons) are of particular interest to aerobiology1. Humans may become rapidly infected with some of these pathogens by large respiratory droplets or small-particle nuclei and easily spread these pathogens to others through salivary secretions, coughing, and sneezing2. In the US biodefense community, high-consequence pathogens (e.g., filoviruses or other NIAID Category A-C Priority Pathogens and CDC Bioterrorism Agents) are the focus of aerosol research programs due to high lethality of associated infections3,4. Significant scientific strides within the aerobiology field have been made within the past decade due to technological advances in aerosol equipment and high containment facilities5,6. Research at the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIH/NIAID), Integrated Research Facility at Fort Detrick located in Frederick, MD, USA (IRF-Frederick) focuses on high-consequence emerging pathogens that require animal biosafety level 4 (ABSL-4) containment. The overall mission of the IRF-Frederick is to evaluate and facilitate the development of candidate vaccines and therapeutics (medical countermeasures).
Research with high-consequence pathogens at the IRF-Frederick is governed by stringent biosafety and animal care and use requirements. These requirements are outlined in the Biosafety in Microbiological and Biomedical Laboratories (BMBL) manual7 and the federal animal welfare regulations. These necessary requirements may restrict the type of research that can be performed and impact overall study design. As we previously described in this journal, all research conducted in an ABSL-4 environment requires particular caution, highly specialized training, and a robust and redundant facility infrastructure8,9.
Entry into the IRF-Frederick ABSL-4 suit laboratory requires donning a positive-pressure encapsulating suit8. Positive-pressure encapsulating suits are not required for entering the ABSL-4 cabinet laboratory. Donning a scrub suit, rubber or nitrile gloves, and close-toed shoes is appropriate when manipulating Risk Group 4 infectious material within a certified Class III biosafety Cabinet (BSC) in an ABSL-4 cabinet laboratory7.
At the IRF-Frederick, aerosol equipment is engineered, assembled, and maintained in two hermetically sealed, stainless steel, air-tight, negative-pressure Class III BSCs, Figure 1. The IRF-Frederick Aerobiology Core employs an automated aerosol management Platform (AAMP) to control and monitor aerosol experimentation within these BSCs, Figure 2. A previous publication outlined the specific functions of the Class III BSCs at the IRF-Frederick and the connection to the suit laboratory via a pass-through port5. The procedure of preparing the Class III BSC prior to experimentation is specific to the IRF. Other Class III BSCs used at other institutions function similarly to the Class III BSC in use at the IRF, but may have different mechanisms for transport, access, or docking.
To further understand how high-consequence pathogens remain infectious and spread through aerosol transmission, safe aerobiological experimentation must be conducted in these Class III BSCs according to a specific workflow procedure. Researchers have been carefully and thoroughly trained to ensure this workflow is followed in a safe and consistent manner. Prior to nonhuman primate (NHP) aerosol challenge, several aerosol characterization or sham aerosol runs are performed to test the stability and viability of an agent when in aerosol form. The aerosol characterization process mimics the actual aerosol challenge, and the researcher evaluates the variables associated with aerosol studies.
Another part of the workflow is to record physical manipulations, administration or anesthetics or other agents, or routine procedures on charts for each NHP. These subject charts are analyzed thoroughly to ensure procedural consistency and standardization. Subjects are anesthetized prior to aerosol exposure. Example anesthetics include tiletamine/zolazepam, ketamine/acepromazine, and ketamine. Anesthetics are chosen based on minimizing respiratory suppression and promotion of controlled, steady-state breathing. Additional anesthesia supplies are maintained in the animal procedure rooms and transported on the transfer cart with the NHP to the aerobiology ABSL-4 cabinet laboratory.
Within the ABSL-4 suit laboratory, NHPs undergo plethysmography via one of two methods (i.e., head-out plethysmography, respiratory inductive plethysmography [RIP]) to determine inspiratory tidal volume and breathing rate changes10-12. These derived parameters are used for accurate calculation of the estimated inhaled dose of the pathogen immediately prior to or during an aerosol exposure. Head-out plethysmography uses a long, cylindrical chamber that houses the NHP13. The pressure drop created when an animal is in the cylinder is captured by a pneumotachograph, relayed to the amplifier, processed by the alternating current/direct current converter, and integrated into the software to derive the above pulmonary parameters. RIP uses sensors made of inductive coiled copper wires that are embedded in elastic bands around the subject&#39;s chest and abdomen11,12. An inductive-capacitor generates a magnetic field in the sensor. Breathing changes the magnetic field, and the resulting voltage changes are relayed from a transmitter next to the elastic band to a receiver in the computer via short-wavelength ultra-high frequency radio waves. Dedicated software determines breathing rate and tidal volume from total thoracic displacement.
The minute volume (MV) obtained through plethysmography is used in the calculation of the estimated inhaled dose (D). In generating and sampling an aerosol, the aerosol concentration (AC) is calculated by multiplying the biosampler concentration (BC) by the volume of media (V) and dividing by result of multiplying the flow rate of the biosampler (FL) by the exposure time (T). The simplified formula is represented as AC = BC x V &#247; FL x T. In turn, for the actual aerosol challenge in NHPs, D is calculated by multiplying AC by MV and the exposure duration (time = T). The simplified formula is represented as D = AC x MV x T.
The purpose of this article is to visually demonstrate the entire aerosol challenge procedure using NHPs from two viewpoints, the ABSL-4 suit laboratory side and the ABSL-4 cabinet laboratory side. Although these procedures may be general in nature for several practices mentioned, they are specific to the IRF-Frederick Aerobiology Core and represent the actual practices used at this institution. This article focuses on the biosafety procedures needed to safely perform an aerosol challenge, not the actual aerosol challenge itself. In these procedures, we are using a dummy subject to show biosafety practices, due to the risk associated with anesthetizing an NHP. However, the process of performing an aerosol challenge is written in a general way because the procedure is the same regardless of high-consequence pathogen used. We aim to enhance knowledge and understanding of scientists about the rigors of conducting aerosol studies of high consequence pathogens under maximum containment conditions.

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			<td height="21" style="height:21px;width:340px;">Micro-Chem Plus</td>
			<td style="width:239px;">National Chemical Laboratories</td>
			<td style="width:139px;">255</td>
			<td style="width:200px;"></td>
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			<td height="21" style="height:21px;">Ethanol&#160;</td>
			<td>Fisher&#160;</td>
			<td>BP2818500</td>
			<td></td>
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			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>441244</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Class III BSC</td>
			<td>Germfree</td>
			<td>DGB-10</td>
			<td></td>
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			<td height="21" style="height:21px;">Integrated BSC gloves</td>
			<td>Piercan</td>
			<td>10UY2032-9</td>
			<td></td>
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			<td height="21" style="height:21px;">Aerosol Management Platform (AeroMP)</td>
			<td>Biaera Technologies</td>
			<td>NA</td>
			<td></td>
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			<td height="21" style="height:21px;">Head-out plethysmography</td>
			<td>Buxco/Data Sciences International</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Respriatory inductive plethysmography</td>
			<td>Data Sciences International</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centered flow tangential aerosol generator (CenTAG)</td>
			<td>CH Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collison nebulizer</td>
			<td>BGI Inc.&#160;</td>
			<td>CN25</td>
			<td></td>
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			<td height="21" style="height:21px;">Autoclave</td>
			<td>Getinge</td>
			<td>GEB 2404 AMB-2</td>
			<td></td>
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			<td height="21" style="height:21px;">Sperian positive-pressure suit</td>
			<td>Honeywell Safety Products</td>
			<td>BSL 4-2</td>
			<td></td>
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			<td height="21" style="height:21px;">Outer suit gloves (latex, Ansell Canners and Handlers)</td>
			<td>Fisher</td>
			<td style="width:139px;">19-019-601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Outer suit gloves (nitrile/rubber, MAPA)</td>
			<td>Fisher</td>
			<td>2MYU1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scrubs</td>
			<td>Cintas</td>
			<td>60975/60976</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Socks</td>
			<td>Cintas</td>
			<td>944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Duct tape</td>
			<td>Pack-N-Tape</td>
			<td>51131069695</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Towels</td>
			<td>Cintas</td>
			<td>2720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O-rings</td>
			<td>O-ring warehouse</td>
			<td>AS568-343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Overshoes</td>
			<td>Amazon</td>
			<td>B0034KZE22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zip lube</td>
			<td>Amazon</td>
			<td>B000GKBEJA</td>
			<td></td>
		</tr>
	</tbody>
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safety precautions and operating procedures in an (a)bsl-4 laboratory: 3. aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

The Class III biosafety cabinet (BSC) is a hermetically sealed stainless steel cabinet containing an ABSL-4 environment under negative pressure within an ABSL-4 cabinet laboratory (Figure 1). Materials can be introduced into the BSC by staff working in the ABSL-4 cabinet laboratory through an under-cabinet-mounted stainless steel tank (commonly referred to as a &#34;dunk tank&#34; in ABSL-4 or BSL-4 settings) containing a 5% dual quaternary ammonium (n-alkyl dimethyl benz...

Watch this Scientific Journal Video about Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology at JoVE.com

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

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