Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways

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Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways S. Kabilan a,n, S.R. Suffield a, K.P. Recknagle a, R.E. Jacob a, D.R. Einstein a, A.P. Kuprat a, J.P. Carson b, S.M Colby a, J.H. Saunders c, S.A. Hines c, J.G. Teeguarden a, T.M. Straub a, M. Moe d, S.C. Taft e, R.A. Corley a a

Pacific Northwest National Laboratory, 902 Battelle Boulevard, P.O. Box 999, MSIN J4-16, Richland, WA 99352, United States Texas Advanced Computing Center, Austin, TX 78758, United States c Battelle, 505 King Avenue, Columbus, OH 43201, United States d Department of Homeland Security, Science and Technology Directorate, Washington, DC 20528, United States e U.S. Environmental Protection Agency, National Homeland Security Research Center, Threat and Consequence Assessment Division, Cincinnati, OH 45268, United States b

a r t i c l e i n f o

Keywords: Three-dimensional computational fluid dynamics Particle deposition New Zealand white rabbit Human Bacillus anthracis Lung

n

abstract Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived respectively from computed tomography (CT) and mCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation–exhalation breathing conditions using average species-specific minute volumes. Two different exposure scenarios were modeled in the rabbit based upon experimental inhalation studies. For comparison, human simulations were conducted at the highest exposure concentration used during the rabbit experimental exposures. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the nasal sinus compared to the human at the same air concentration of anthrax spores. In contrast, higher spore deposition was predicted in the lower conducting airways of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in speciesspecific anatomy and physiology for deposition. & 2016 Published by Elsevier Ltd.

Corresponding author. Tel.: þ 1 509 371 6918. E-mail address: [email protected] (S. Kabilan).

http://dx.doi.org/10.1016/j.jaerosci.2016.01.011 0021-8502/& 2016 Published by Elsevier Ltd.

Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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1. Introduction Inhalation anthrax is an infectious and potentially fatal disease caused by Bacillus anthracis, a gram-positive, endosporeforming, rod-shaped bacterium (Grinberg, Abramova, Yampolskaya, Walker, & Smith, 2001; Plotkin, Brachman, Utell, Bumford, & Atchison, 2002). Exposure and potential disease in humans can occur cutaneously, via ingestion, or, most lethally, via inhalation (Frazier, Franks, & Galvin, 2006). Inhalation anthrax results from the inhalation of B. anthracis spores that deposit in the respiratory tract thereby gaining access to favorable physiological locations for germination and initiation of infection. Concern about the use of B. anthracis as a bioterrorist agent was heightened when 22 infections and five fatalities were reported in 2001 after spore-laced letters were sent through the U.S. postal system (Dewan et al., 2001). Hence, priority has been placed on developing computational models of inhaled B. anthracis spores to mechanistically model anthrax disease and to derive dose-response relationships for use in humans. Mechanistic models are available for the rabbit and the human that describe selected biological events associated with exposure, disease progression, and host responses from exposure to B. anthracis spores (Brookmeyer & Blades, 2002; Day, Friedman, & Schlesinger, 2011; Gutting, 2014; Wilkening, 2008), though a comprehensive model is yet to be developed for the entire disease process. Mechanistic models that evaluate early disease events immediately after inhalation of B. anthracis spores incorporate consideration (either implicitly or explicitly) of the respiratory tract region(s) assumed responsible for the initiation of infection. Current conceptual models for the initiation of B. anthracis infection include the Trojan horse model (Guidi-Rontani, 2002) that hypothesizes macrophages are primarily involved in translocation of spores across the alveolar barrier and into the lymphatic system where germination and proliferation can occur versus the jailbreak model (Weiner & Glomski, 2012) that identifies multiple respiratory and/or oral-pharyngeal mucosal-associated lymphoid tissues as having a potential role in local germination and release of toxins and proteases that results in a breakdown in the epithelial barrier allowing non-host cell transport of spores and bacteria into the lymphatic system. Given the region-specific nature of the initiation of infection, approaches to mechanistically model inhalation anthrax must link atmospheric concentrations of anthrax spores to inhaled “doses” deposited in each region of the respiratory system that contributes to disease onset and progression. Accordingly, a key element in mechanistic modeling and the development of dose-response relationships for inhalation anthrax is the appropriate mathematical definition of dose, also termed the dose metric, that can be compared across exposure conditions, species, and respiratory regions. It is well known that particle size, shape, and density along with biological differences in anatomy and physiology determines the region(s) of deposition in the lung (Stahlhofen, Rudolf, & James, 1989; Thomas, 2013). Relative to larger particle sizes, respirable particles have been associated in multiple animal models with an increased hazard of inhalation anthrax as early as the 1950s (Druett, Henderson, Packman, & Peacock, 1953). Using this knowledge, an inhaled dose expression of the dose metric was utilized, but adjustments were sometimes made in the measurement of exposure dose to count only those particles in the 1–5 or 10 mm size range (e.g., o 5 mm in Brachman, Kaufman, & Dalldorf, 1966). However, more current data has demonstrated that there is not a strict size cutoff for pulmonary deposition as particles larger than the respirable range may be associated with deposition in this lung region albeit at significantly lesser levels (U.S. Environmental Protection Agency, 1994). Given these factors, the availability of deposition data should drive the selection of a deposited dose over an inhaled dose if sufficient data are available (Gutting et al., 2008; U.S. Environmental Protection Agency, 2010). Given the scarcity of quantitative human exposure data for inhalation anthrax, animal data have been a critical component in the development of mechanistic models and dose–response relationships. The rabbit is a commonly used animal model for inhalation anthrax research. Benefits of the rabbit animal model include a history of use in anthrax natural history studies, end-stage anthrax pathology very similar to the human and the nonhuman primate, and current use in conjunction with anthrax vaccine efficacy and medical countermeasure studies (Coleman, Thran, Morse, Hugh-Jones, & Massulik, 2008; Gutting et al., 2012; Pitt et al., 2001; Yee, Hatkin, Dyer, Orr, & Pitt, 2010; Zaucha, Pitt, Estep, Ivins, & Friedlander, 1998). However, interspecies differences between animal models and the human necessitate use of an extrapolation process prior to the direct use of animal data for the human to account for known contributors to response differences. For inhaled exposure of chemicals, varying particle deposition patterns, clearance mechanisms, and biological responses have been implicated in causing interspecies differences in response (Corley et al., 2012; Kabilan, Lin, & Hoffman, 2007). In the case of modeling B. anthracis exposure and development of inhalation anthrax, these same elements are important but also currently represent areas of considerable uncertainty in the microbial pathogen risk assessment process (Coleman et al., 2008). The development of robust particle deposition data in the rabbit and the human would greatly contribute to advances in the dosimetric extrapolation process for available dose-response data. Particle deposition data for the rabbit are available describing whole lung or region-specific values (Gutting et al., 2013; Gutting et al., 2012; Raabe, Al-Bayati, Teague, & Rasolt, 1988) (Table 1). However, the reliability and precision of the measurement techniques raises potential issues for their application in modeling. Potential biases in the identified measurement approaches are described in Table 1. More recent data specific to B. anthracis spore deposition that utilizes bronchoalveolar lavage may undercount deposited particles due to potential translocation within the lungs (Gutting et al., 2012). Researchers modeling inhalation anthrax have used a number of deposition models to estimate deposited spore concentration from exposure concentration in the lung (Gutting et al., 2008). These mathematical models include the semiempirical compartment model (International Commission on Radiological Protectio, 1994), trumpet model (Choi & Kim, 2007), single-path model (Yeh & Schum, 1980), multiple-path model (Anjilvel & Asgharian, 1995), and stochastic asymmetric multiple-path model (Koblinger & Hofmann, 1990). In the case of the semi-empirical compartment model, the respiratory Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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Table 1 Published deposition values for the rabbit using direct measurement approaches. Study

Reported value

Measurement approach and material

Potential bias in outputs

Gutting et al. (2013)

Pooled value of 4.63% from two data set values: 4.33% ( 7 2.2%) and 4.93% ( 7 0.8%), represents whole lung deposition

Potential for underestimation of deposition if epithelial cell internalization of deposited particles is rapid (e.g., see Jenkins and Xu (2013) data in mouse animal model)

Gutting et al. (2012)

3.077 0.9% and 1.3370.2%, represents whole lung deposition

Homogenization of New Zealand white rabbit lung tissue and extrapolation to the whole lung after inhalation exposure to B. anthracis spores, particle size MMAD of 1.0 70.3 mm Bronchoalveolar lavage to wash out deposited B. anthracis spores in New Zealand white rabbit, particle size MMAD of 1.0 7 0.3 mm

Raabe et al. (1988) Note: Raabe et al. (1988) data were the basis for U.S. EPA’s RDDR model as described in U.S. Environmental Protection Agency (1994)

Ranging from 6.6 7 0.6% at 0.97 mm to 1.17 0.2% at 4.86 mma, pulmonary deposition only

Measurement of deposition to pulmonary region of the rabbit after inhalation of monodisperse 169Yb aluminosilicate aerosol with aerodynamic resistance diameters of particles ranging from 0.18 to 8.65 mm

Deposited doses reported from bronchoalveolar lavage may be biased low if inability to wash out all deposited spores or rapid transport across epithelial cell lining takes place (Gutting et al., 2012) Use of Guyton’s formula to estimate minute volume for calculation of deposition would bias results if actual animal inhalation rate differed (Raabe et al., 1988)

RDDR – regional deposited dose ratio; MMAD – mass median aerodynamic diameter. a Aerodynamic resistance diameter measurement.

tract is treated as a series of filters through which the inhaled particles pass during inhalation and back again during exhalation (International Commission on Radiological Protectio, 1994). The characteristic parameters that govern these filters are fraction of the tidal air that reaches an individual filter and the deposition efficiency of that filter. Though these models are based on measured species-specific airway morphology, they are greatly simplified and assume homogenous deposition within these regions. The trumpet models assume the airway system to be a symmetric, one-dimensional, variable cross-sectional channel where the cross-sections are functions of generation number (Choi & Kim, 2007). A significant limitation of this approach is the simplistic airway geometry without any internal airway structure and the inability to simulate asymmetric effects of airway geometry. The basis of the single path model is that all pathways of an inhaled particle from the trachea to the terminal branches in a symmetric lung model are identical and thus can be represented by a single path (Yeh & Schum, 1980). Because of the symmetric branching, the inhaled airflow and particles are equally distributed among all airways in a given airway generation. Due to the assumption of simplistic lung structure, these models cannot be used to predict deposition in monopodial or variable lung structures. Multiple-path models are more realistic than the single-path models because they are based on actual measurements of airways and their associated branching structure and can also represent an asymmetric branching lung (Anjilvel & Asgharian, 1995). Their main limitation is the lack of availability of species-specific airway morphometric data, especially for the upper conducting airways. This limitation is overcome in the stochastic multiple-path model by populating the lung with asymmetrically branching airways and stochastically tracking particles (Koblinger & Hofmann, 1990). While modeling the dose-response relationship for inhalation anthrax, these models help predict delivered dose from exposure concentrations in the air, which reduces uncertainties during low-dose and cross-species extrapolation. But, the delivered dose is a function of both individual and species-specific anatomy and physiology. Three-dimensional (3D) computational models of particulate deposition in the lung have become highly developed (Kleinstreuer, Zhang, & Li, 2008; Rostami, 2009) and provide the ability to track patterns of deposition through the pulmonary system as a function of the morphology, breathing parameters, and particle characteristics. While solutions for the full lung require simplifications to prevent excessive computational time, solutions for the upper branches, using representative pulmonary morphology obtained from casts or scans, are quite feasible. Advances in image processing and meshing techniques, coupled with robust solvers and the prevalence of computational clusters, have greatly reduced the time required to generate these models and obtain solutions (Corley et al., 2012; Kuprat, Kabilan, Carson, Corley, & Einstein, 2013). Given the critical importance of deposition data to further mechanistic and dose–response modeling of inhalation anthrax, there is a clear need to develop robust estimates of B. anthracis spore deposition for the human and the rabbit that are representative of the species-specific, 3D airway geometries. In the current work, physiologically realistic, image-based 3D airway geometries of the human and rabbit were used with computational fluid dynamics (CFD) airflow modeling coupled with Lagrangian particle tracking methods to predict the inhalation and deposition patterns of B. anthracis spores during transient breathing. The rabbit and the human geometries used in the current study provide highly detailed geometries with greater resolution than previously published imagingPlease cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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based models. To the best of our knowledge, this is the first-ever 3D model of B. anthracis spore deposition in speciesspecific imaging based geometry.

2. Methods 2.1. Rabbit Imaging Six Male New Zealand white rabbits weighing 3.5–3.8 kg were obtained to develop protocols for imaging the upper and lower respiratory airways. Each animal was humanely sacrificed via intravenous injection of  150 mg/kg pentobarbital, and the pharynx, larynx, and trachea imaged at 100 micrometers resolution using computed tomography (CT) (XT H 225/320 LC, Nikon Metrology, Inc.). The intact, dead rabbit was positioned to imitate a natural sitting posture used in prior acute studies. However, due to post mortem settling during the long (  1 h) acquisition time, images of the nasal airways were slightly blurred and were not suitable for segmentation of highly intricate and detailed structures in the sinus. Therefore, to acquire high-resolution images of the nares and sinus, the heads were removed at the neck above the larynx and imaged at 50 micrometers resolution in a μCT scanner (GE eXplore CT-120). Two adjacent scans were required per specimen because sinus dimensions exceeded the field of view of the scanner. As shown previously (Corley et al., 2012), high-resolution imaging of tracheal-bronchial airways can be enhanced by using lung casts. An in situ lung airway casting protocol was therefore developed with a hard-curing casting agent that preserves the branching structure and branch angles based on previous airway casting methods developed for rats (Jacob, Colby, Kabilan, Einstein, & Carson, 2013). Once the lung casting protocol for the rabbit was developed, four additional male New Zealand white rabbits weighing 3.0– 3.3 kg were acquired. After humane sacrifice the fur on the neck and thorax was clipped, and the skin covering the ribcage was removed. The trachea was exposed just below the larynx, and a 6.4 mm outside diameter tube was securely tied into the trachea using 3-O suture. Slits were then cut between several ribs to allow for lung collapse. The lungs were then inflated to 25 cmH2O with saline through a 3-way valve. The casting agent was mixed, consisting of a 10:1 mixture (by weight) of 2-Tons epoxy (ITW Devcon, Danvers, MA) and 200 Fluids, 20 CST (Dow Corning, Midland, MI). After thorough stirring for 1 minute, the mixture was manually spun in a  70 cm radius centrifuge at  3 Hz for 2 min to extract air bubbles – the mixture’s viscosity and short hardening time made vacuum degassing impractical. The mixture was then drawn into a 20 mL syringe. The syringe was placed on a syringe pump, attached to the valve, and 8.5-9.0 mL of the mixture was injected at a rate of 200 mL/h. The injected volume was determined during the preliminary casting work. Any large air bubbles that became trapped in the dead space of the valve were drawn from the tubing using a 30-gauge needle and syringe. After filling, the cast was allowed to cure for  18–24 h while the animal was refrigerated. The lungs were then removed from the chest cavity, and the lung tissue was digested away in household bleach ( 4 h). After thorough rinsing, the rabbit casts were imaged at 50 micrometers resolution. As with nasal airway imaging, two adjacent mCT scans were required per cast because of scanner field of view limitations. The results from the highest quality rabbit cast was used in CFD modeling. 2.2. Human imaging The human respiratory geometry was based on multi-slice CT imaging of the head and torso of a healthy 35-year-old male volunteer weighing 150 lbs. and 67 in. tall. A GE Light Speed Discovery CT750 was used to produce an image volume size of 512  512  960 with a resolution of 0.7  0.7  0.5 mm in the x, y and z dimensions. The field of view was 36.0  36.0  48.0 cm. 2.3. Airway segmentation Segmentation is used to delineate a particular feature in an image. Airway segmentation was performed on the image datasets to facilitate volume meshing, and CFD modeling of the airway. Segmentation of the human model was applied as described in Corley et al. (2012). Based on the work of Stanescu, Clement, Pattijn, and van de Woestijne (1972), the larynx for the human model was widened from the as-imaged supine breath-hold position to a fully open geometry that occurs during inhalation in an upright posture. For the current study, human simulations were designed for nasal breathing since normal human respiration at rest is through the nasal route (Swift & Proctor, 1977); thus, the oral cavity and oropharynx were not included in the 3D model. Two different methodologies were followed for the segmentation of the rabbit imaging data. The upper airways and the lung cast data were segmented as described in Carson et al. (2010) and Timchalk, Trease, Trease, Minard, and Corley (2001), respectively. Briefly, for the upper airways, an intensity-based threshold was set without pre-filtering, followed by visual validation and repair. For the lung cast data, edge-preserving hybrid median filters and background normalization were used to remove both high and low frequency noise, respectively. Then, intensity thresholds followed by manual inspection were used to identify the lung cast boundary. Finally, the lung cast topology was semi-automatically validated and repaired to ensure no loops of overlapping lung airways—caused by close proximity of smaller airways that were not resolvable by the imaging system—remained. Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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2.4. Mesh generation Isosurfaces were extracted by applying a variant of the marching cubes algorithm (Lorensen & Cline, 1987) to the segmented CT lung image in the case of the human dataset and to the segmented mCT images of airway casts in the case of the rabbit. Once the isosurfaces were extracted, the centerline of the triangulated lung surface of each species was decomposed into a simplified but topologically correct skeleton that formed the basic data structure for all morphometric measurements, manipulations, and analyses. During mesh generation, the centerline data was used to automatically truncate lung geometries and introduce boundary facets at a user-specified generation or airway diameter (Jiao, Einstein, & Dyedov, 2010). To maximize the number of conducting airway generations included in the rabbit model while excluding alveolar structures in the 3D geometry, a cut-off diameter of 500 μm was used to define bronchiolar airways. This cutoff in airway diameter resulted in a lung model that was made up of 3857 airways with 2878 terminal outlets, Since the human data set was of lower-resolution, all segmented airways with 272 terminal outlets were utilized in the model. The pulmonary airways with truncated outlets were then united with the similarly extracted surfaces for the upper respiratory tract for each species using MAGICS™ (Ver. 16.02, Materialise, Plymouth, MI). To compute the deposition efficiencies, the respiratory geometry of the rabbit and the human was annotated in MAGICS based on anatomical regions. The models consisted of the nose, pharynx, larynx, lower trachea, and bronchi and bronchioles regions as shown in Fig. 1. Hybrid prism/polyhedral volume meshes were generated in STAR-CCMþ (Version 8.02, CD-Adapco, Meville, NY). The isosurfaces from final segmented airways were imported, remeshed and hybrid volume meshes were generated using the standard polyhedral and prism layer meshing utility in STAR-CCMþ. The tightly packed boundary layer consisted of

Fig. 1. Annotations of human and rabbit respiratory tract based on anatomical regions for calculating deposition efficiency.

Fig. 2. Hybrid-poly mesh generated in STARCCM for the human model showing a mesh along the cross-sectional plane AA0 and the inset showing close-up of the prismatic boundary layer.

Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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prismatic elements to accurately capture boundary layer formation and deposition of spores at the airway walls. Polyhedral mesh elements constituted the core of the volume mesh. To assess the sensitivity of the mesh size on airflow and spore deposition, the total number of elements for the human model was increased by 40% and the dimensionless wall distance (yþ ) was monitored. Between the two meshes, among all the anatomical regions, the highest difference in spore deposition was 0.39% in the nasal region and the yþ was less than one for both meshes, indicating that the original mesh is adequate to resolve airflow near the wall regions. The final mesh for the human model consisted of 5,751,626 nodes and 2,550,285 polyhedral elements. Similarly, the rabbit mesh consisted of 36,232,173 nodes and 16,060,891 polyhedral elements (Fig. 2). 2.5. Airflow CFD airflow simulations were performed using STAR-CCMþ. The airflow predictions were based upon the turbulent 3D, incompressible mass and Reynolds-Averaged Navier–Stokes (RANS) momentum equations: ∂uj ¼0 ∂xj

ρ

ð1Þ

  ∂ui ∂u ∂p ∂2 ui ∂ þ u j i ¼  þ ρg i þ μ  ρu 0 u 0 ∂xi ∂t ∂xj ∂xj ∂xj ∂xj i j

ð2Þ

In the above expressions t is time, ui or uj is the mean fluid velocity component in coordinate direction xi (i¼1, 2, 3), repeated subscripts denote summation, g i are components of gravity vector, ρ is the gas density, p is the pressure, and m is the dynamic viscosity. The u0i and u0j in the right-most term of Eq. (2) are velocity fluctuations about the mean velocity, and the overbar indicates the time averaging of the products of these fluctuations. This term represents the additional Reynolds stresses due to turbulent motion. These are linked to the mean velocity via the turbulence model being used. In the present work, the generation and dissipation of turbulence was accounted for using the SST k-omega model as described in the STAR-CMMþ User Guide (CD-Adapco 2013). The model performs well for swirling flows such as those produced in the nasal turbinates and larynx. The model was formulated by blending the standard k-omega near the surface with a transformed kepsilon model in the bulk flow. For all CFD simulations, air at room temperature was considered to be the working fluid, with a density of 1.0 kg/m3 and a dynamic viscosity of 1.5025  10  5 m2/s. The inlets for both species were prescribed a “time dependent flow rate” boundary condition in which the CFD code adjusted the magnitude of the inlet velocity to match the user specified volumetric flow rate and breathing profile. A minute volume of 7.4 l/min and breathing frequency of 20 breaths per minute (BPM), approximating resting breathing conditions, was used for the human (International Commission on Radiological Protectio, 1994). For the rabbit, a minute volume of 1.745 l/min was used, computed using the sampling time period and accumulated tidal volume from telemetric data generated during the B. anthracis inhalation study using the New Zealand white rabbit described in U.S. Environmental Protection Agency (2011). A breathing frequency of 80 BPM was assumed (Maskrey & Nicol, 1980). A simple sine wave based on the minute volume and breathing frequency was an idealized assumption to mimic inhalation and exhalation phases of the breathing cycle for each species. A zero-pressure boundary condition was considered adequate for the outlets because of the relatively uniform size of all terminal outlets. A no-slip wall condition was applied to the remaining airway boundaries, which were assumed to be rigid and impermeable. These models are available to readers through the journal supplemental data or by contacting the authors. 2.6. Spore simulations Two different B. anthracis exposure scenarios were modeled to mimic the exposure of rabbits described in U.S. Environmental Protection Agency (2011). Cases 1 and 2 (Table 2) reported herein were derived from the rabbit inhalation and dose data associated with Groups 3, 4, 5, and 6, respectively, as described by the inhaled dose data in Table 5 of U.S. Environmental Protection Agency (2011). Using methods described in Taft and Hines (2012) for benchmark dose modeling of microbial pathogens, the calculated BMD50 (benchmark dose at 50% extra risk) and BMDL50 (benchmark dose, lower 95% confidence limit at 50% extra risk) values were Table 2 Experimental data from the acute rabbit exposure study conducted by EPA and computed cumulative concentration used during modeling for Case 1 and Case 2. The highest exposure concentration (Case 1) was adapted to model the human exposure scenario. Case

MMADa (μM)

GSDa

Accumulated tidal volumea (m3)

Actual concentrationa (spores/m3)

Cumulative concentrationb (spores/m3)

Case 1 Case 2

1.12 0.92

1.31 1.57

1.19  10  02 1.31  10  02

7.27  1008 1.96  1005

3.97  1011 1.18  1008

Note GSD – geometric standard deviation. Accumulated tidal volume:total volume inhaled over exposure time. Tidal volume for rabbit¼2.18  10  05 m3. a Experimental data. b Computed; cumulative concentration used during modeling (spores/m3) ¼actual concentration  total cumulative tidal volume/tidal volume.

Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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52,000 CFU (colony-forming units) and 13,000 CFU, respectively (U.S. Environmental Protection Agency, 2012). The calculated BMD10 (benchmark dose at 10% extra risk) and BMDL10 (benchmark dose, lower 95% confidence limit at 10% extra risk) values were 5700 CFU and 1400 CFU (U.S. Environmental Protection Agency, 2012). One intended application of the CFD modeling is the development of a dosimetric adjustment factor for spore deposition in the rabbit relative to the human. Note: The word “particle” and “spore” are interchangeably used in the reminder of the article. The Lagrangian particle approach used in these simulations tracks each spore, properly representing the inherent (natural) randomness (a stochastic process) of spore deposition. A very large number of spores must therefore be tracked to observe the trends in deposition over the natural “noise” from stochasticity in spore deposition. Since the concentrations of B. anthracis spores used in the acute inhalation studies conducted by EPA were ultra-low (e.g. o4  1011 spores/m3), a large number of breaths, which are computationally intensive, would have been required to generate a sufficient number of deposited spores to achieve stability in the simulation results. Since spores were assumed to be non-interacting in the simulations and breathing characteristics were invariant breath to breath, it is possible to accurately simulate the sum of many breaths at a low spore concentration using a single breath of a much higher spore concentration. Therefore, a single full breath cycle consisting of a complete inhalation and exhalation phase was used to simulate the cumulative concentration of spores inhaled over the duration of the acute inhalation exposure (Table 2). Accordingly, the model assumes no exposure concentration-based dose-dependency in deposition pattern. The highest calculated cumulative exposure concentration used during the acute rabbit inhalation experiment was adapted to model the human exposure scenario. Based upon the assumptions described below, this approach is demonstrably equivalent to the much more computationally intensive multiple breath approach. The standard Lagrangian dispersed two-phase flow and particle-tracking algorithm in STARCCM þ was utilized with the following common assumptions: 1. One-way coupling – since the spores are micron sized and the mass loading at the inlet is low, the assumption that the spore does not affect airflows and airflow patterns was applied (Zhang & Kleinstreuer, 2004). 2. Non-interaction between B. anthracis spores – the simulations do not include spore-spore collisions or agglomeration; a reasonable assumption given the ultra-low concentrations used in the simulations and animal experiments. 3. Mono-dispersity – Due to the low spore concentration at the inlet, it was assumed that the B. anthracis spore diameter is constant and remains a constant throughout the simulation. 4. Equivalent aerodynamically spherical spores with a density of 1 g/cm3. 5. Spores that exited the 3D outlets of the bronchial airways of the 3D lung were assumed to deposit in the “deep lung” compartment of our simulation results. There is currently no method available for reintroducing particles that left the 3D terminal airways that remain suspended in airways of the deep lung back into the 3D domain during exhalation. Thus, “deep lung” deposition results from these simulations represent an upper bound prediction of spores that actually reach the gas exchange region of the lung in each species. 6. No-slip boundary condition for spores at the airway wall. In this boundary condition, once the spore comes in contact with the airway wall, it is assumed to adhere to the wall and is not allowed to slide along the wall or become reintroduced into the flow field at a later time point. 7. Due to the micron-size of the spores, Brownian force was neglected. Therefore, drag and gravity were the only forces assumed to be acting on the spores. The direction of the gravity vector was such that it mimics the sitting position in the rabbit and upright standing position in the human. Electrostatic charge is presumed to be negligible and was not considered. 8. The airway is considered rigid with smooth walls. Due to unavailability of data to prescribe moving airway boundaries, most published 3D CFD models of particle deposition have used rigid walls (Kleinstreuer et al., 2008). This assumption is reasonable since most airway geometry captured by imaging modality is in the proximal lung where airway movement is relatively less and reflects the current state of the science. Lastly, all calculations of spore deposition across species represent initial deposition patterns only; no clearance mechanisms for deposited spores were included in the current model as time beyond an inhalation and exhalation is not included in the model.

3. Approvals The Institutional Animal Care and Use Committee of PNNL approved all animal work conducted under this study. The deidentified human imaging data were obtained under a separate project at the University of Washington (UW), Seattle and was approved by the Institutional Review Board at UW and PNNL and informed consent was obtained for experimentation with human subjects.

Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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Table 3 Deposition efficiencies for different annotated regions in the rabbit and the human nasal model. The spores that escaped through the outlets of the 3D geometry were included in the “deep lung” compartment. There are currently no methods for re-introducing spores that did not deposit in the deep lung back into each airway outlet of the 3D model during the exhalation phase of the breathing cycle. Case

Location

% Deposition based on exposure Rabbit

Human

Case 1

Nose Pharynx Larynx Trachea Bronchi and Bronchioles Deep lung (not modeled)

12.61 0.03 0.13 0.07 1.44 54.34

3.21 0.12 0.33 0.01 5.70 62.08

Case 2

Nose Pharynx Larynx Trachea Bronchi and bronchioles Deep lung (not modeled)

7.05 0.01 0.16 0.06 1.49 58.94

– – – – – –

The total spore deposition for the rabbit and the human was 68.62% and 71.45%, respectively for Case 1.

Table 4 Compartment specific spore count and compartment area normalized spore deposition for the rabbit and human model based on the % deposition reported in Table 3 for the actual highest concentration used in the acute experimental study (Table 2) conducted by EPA. The human dose is greater than rabbit for all compartments except the trachea. Case

Case1

Concentration (spores/m3)

7.27  1008

Region

Nose Pharynx Larynx Trachea Bronchi and bronchioles

Rabbit

Human 2

Total spore count

Dose (spores/cm )

Total spore count

Dose (spores/cm2)

2.00  1003 4.75 2.06  1001 1.11  1001 2.28  1002

1.69  1001 9.42  10  01 5.13 8.10  10  01 3.21

8.63  1003 3.23  1002 8.88  1002 2.69  1001 1.53  1004

3.87  1001 1.11  1001 1.93  1001 5.46  10-01 5.15  1001

Note: Acute experimental spore concentration (C; spores/m3) ¼7.27  1008. Tidal volume (TV; m3): rabbit¼2.18  10  05; human¼ 3.70  10  04. Number of spores per breath (C  TV): rabbit¼ 1.58  1004; human¼ 2.69  1005.

4. Results Table 3 summarizes the region-specific deposition efficiencies after one breathing cycle for the rabbit and the human models exposed to the same B. anthracis spore concentration as identified in Case 1. Case 2 is a lower experimental exposure scenario with slightly smaller MMAD (mass median aerodynamic diameter) spore sizes modeled only in the rabbit. For Case 1 (1.12 mm MMAD, 3.97  1011 spores/m3) in the rabbit model, the nose accounted for 12.61% of inhaled spores, which is the maximum deposition for all of the annotated regions. The deposition efficiencies in the pharynx, larynx and the trachea were 0.03%, 0.13% and 0.07%, respectively. The bronchi and bronchioles region accounted for 1.44%, while 54.34% exited the bronchiolar airways of the 3D and was assumed to represent an upper bound on the percent deposition in the deep lung. Similar to Case 1, the maximum spore deposition within any annotated region for Case 2 (0.92 mm MMAD, 1.18  108 spores/m3) was found to be 7.05% in the nose region. Secondly, the 3D bronchi and bronchioles region accounted for 1.49% of the total exposed spores. The pharynx, larynx and the trachea accounted for 0.01%, 0.16% and 0.06% respectively. Of the total exposure, 58.94% exited the 3D domain and was assumed to represent an upper bound on the percent deposited in the deep lung. The total spore deposition in the rabbit model was 68.62% and 67.71% for Case 1 and Case 2, respectively, with the remainder exhaled or suspended within the 3D domain at the end of the full breathing cycle. The deposition efficiency values represent the percentage deposition based on initial exposure concentration. Unlike the rabbit model, maximum spore deposition was observed in the bronchi and bronchioles region of the human model, which accounted for 5.70% of the total exposed spores over the entire breathing cycle. Secondly, the nose accounted for 3.21% of the exposed concentration. The pharynx, larynx and the trachea accounted for 0.12%, 0.33% and 0.01% respectively. Lastly, the deep lung (spores that exited the bronchiolar airways) accounted for 62.08% of the total exposed spore mass. The total deposition efficiency (assuming all spores reaching the deep lung deposited) in the human model was 71.45% over the full breathing cycle. Table 4 shows total spore deposition for each compartment and compartment specific dose (i.e., spores/surface area of compartment) for the actual experimental exposure concentration (Table 2, Case 1) in the rabbit and human geometry using Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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the predicted regional deposition as reported in Table 3. The human dose is greater than the rabbit dose for all the compartments except the trachea in which the rabbit dose is slightly higher than the human. During the exhalation phase of the breathing cycle in the rabbit model, there was no appreciable difference in the deposition pattern compared to deposition during the inhalation phase. Approximately 1.61% and 1.43% of the total number of exposed B. anthracis spores that remained suspended in the 3D airway lumens at the end of the inhalation phase deposited during the exhalation phase of the breathing cycle for Case 1 and Case 2, respectively. Similar to the rabbit model, there was no noticeable difference in the deposition patterns observed at the end of the exhalation phase in the human model. The exhalation phase accounted for 1.62% of the total number of spores exposed that deposited on the surfaces of the conducting airways while the remainder was exhaled.

5. Discussion In this study, exposure, transport, and deposition of inhaled B. anthracis spores in the rabbit and the human respiratory systems were simulated using realistic cyclic breathing profiles and imaging-based airway geometries. The regional spore deposition in the rabbit and the human respiratory systems agreed well with the local flow characteristics noticed. Since spore-spore interactions were not considered during the Lagrangian tracking simulations and are not generally significant in dilute gas-particle flows, the spore transport is mainly governed by the region-specific airflow characteristics seen in the respiratory airways and convective and gravitational forces applied to the spores. The dependence of spore transport and deposition on local flow characteristics is evident from the noticeable agreement in deposition patterns and visualized highspeed regions at peak inhalation in the human and rabbit model (Fig. 6). In both species, most spores were deposited in the vestibule region as the flow enters the nostrils (Fig. 5). This is due to the locally high-speed airflow that imparts inertia to the spores leading to greater impaction as the flow enters through the narrow airway passage. There was little predicted deposition of spores in the olfactory region for either species. This closely correlates to the flow characteristics seen in the olfactory region of the nose, where it is typical to see very little flow except at the most anterior portion of the olfactory region (Corley et al., 2012). In the pharyngeal and the laryngeal region, the lower relative deposition reported for the rabbit when compared to the human (Table 3) may be related to the smooth curvature of the rabbit pharynx and larynx. The human airway geometry exhibits a nearly 90° turn as airflows transition between the nose and the pharynx, with this geometry having the potential to increase local deposition in the human. However, in the lower tracheal region, due to the curvature in the trachea, the deposition in the rabbit model is relatively higher compared to the human. The effect of tracheal curvature on spore deposition is also evident in Table 4. The dose in the tracheal compartment in the rabbit (no. spores/cm2) is greater than the human tracheal compartment despite lower overall total spore deposition in the trachea compared to the human model. Given that we currently only have one high-quality extended airway model for the rabbit available, a test for statistically significant differences across species and regions was not conducted with these data. 5.1. Model performance evaluation The predicted spore deposition in the upper respiratory track (URT; nose, pharynx and larynx regions in Table 3) and the tracheobronchial (TB; trachea and bronchi and bronchioles region in Table 3) region were compared against experimental

Fig. 3. Comparison of CFD deposition prediction in the URT and TB regions in the rabbit with published experimental data for one-micron aerosol (Raabe et al., 1988) and predictions using the rabbit-specific MPPD model (Asgharian et al., 2015), respectively. Some differences between CFD model predictions to MPPD and experimental measurements is noticeable for the 0.92 μm case due to different minute volume and breathing frequency used in the model.

Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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Fig. 4. Comparison of CFD deposition prediction in the TB for the human model with experimental data of to Kim and Hu (1998). The difference in deposition efficiency between the experiment and model was due to the larger spore size, which would increase inertial impaction, used in the current model.

Fig. 5. (A) Deposited spores in the rabbit and (B) human respiratory models after one breath for Case 1. The insets show deposition in the sinus region.

Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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data for one-micron aerosol deposition from Raabe et al. (Raabe et al., 1988) and predictions using the rabbit-specific Multi Path Particle Dosimetry (MPPD) model derived from the same lung cast used in the CFD model (Asgharian et al., 2015) respectively (Fig. 3). Overall, there is remarkably good agreement between the CFD model predictions, experimental measurements and MPPD model predictions for 1.12 μm MMAD aerosols even though there were differences in breathing frequency and minute volumes across these comparisons. For the slightly smaller 0.92 μm particle size simulations, the CFD model generally under-predicted the deposition efficiencies in the URT compared with both the MPPD model and experimental data. However, in the tracheobronchial region, there was excellent agreement between the CFD and MPPD models with both models under-predicting the experimental data. Kim and Hu (1998) measured regional deposition patterns of inhaled particles in healthy adult male and female human volunteers by means of bolus aerosol delivery technique. The deposition efficiency was measured for 1, 3 and 5 μm at 9, 15 and 30 l/min flow rates. Figure 4 compares CFD model prediction for the tracheobronchial region with the experimental data for 1 μm particle at 9 l/min flow rate. The over prediction was attributed to the comparatively larger spore size (1.12 μm) used in the current model, which would increase inertial impaction at bifurcations during inhalation and the different anatomy of volunteer from which the current CFD model was derived. Swift reported the deposition efficiency in the URT to be negligible for 1 μm particle at 7 l/min unidirectional flow rate in two replicate nasal airway casts (Swift, 1991). At the end of the inhalation phase, the current CFD human model predicts 1.89% deposition in the same region. This difference in deposition could be a result of the wall boundary condition used in the model. In the model, when a spore comes in contact with the wall, it is assumed to be deposited which is different from experimental setup in which the walls were made of plastic that could let particles slide downstream or re-enter the main flow. 5.2. Predicted spore deposition For Case 1 in the rabbit model, 12.61% of the inhaled spores were deposited in the nose, which is nearly four-fold greater than that predicted for the human nose. This is primarily due the greater complexity in nasal airway geometry found in the rabbit. In contrast and despite the fact that the rabbit bronchi and bronchiolar region consisted of far more airways compared to the human model, only 1.44% of the total inhaled spores was deposited compared to 5.70% in the human. This could be attributed to the differences in flow characteristics noticed in the rabbit and the human lung due to different branching pattern (i.e., monopodial branching airway structure in the rabbit with different diameters in daughter airways and bipodial branching pattern in the human with more comparable daughter diameters), and angles of bifurcation (Fig. 6). In the

Fig. 6. (A) Deposited spores in the rabbit and (B) human bronchi and bronchioles region after one breath for Case 1. The insets show a plane extracted at the first bifurcation in the left lung showing contours of velocity magnitude at peak inhalation.

Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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human bronchi and bronchioles region, greater deposition due to impaction were predicted at bifurcations due to the fact that the bifurcations are closer to being perpendicular to the flow field and the angle of bifurcation is greater than that seen in the rabbit lung (Fig. 6(B) inset). Since airways in the rabbit lung branch off the main bronchi at an angle that is less compared to the human biopodial lung branching angle with greater differences in daughter airway diameters that were not perpendicular to the flow field, deposition due to impaction was less when compared to the human model (Fig. 6(A) inset). These types of flow differences in the monopodial and bipodial branching lungs and resulting differences in deposition due to impaction have also been reported by Kabilan et al. (2007). For Case 2, deposition in most annotated regions is similar to Case 1 except for the nasal sinus. This can be attributed to lesser deposition due to impaction in the nasal region due to slightly smaller spore size compared to Case 1 (0.92 vs. 1.12 mm MMAD). As a result of lesser deposition in the upper respiratory tract, the spores that escaped the 3D outlets, which were assumed to have deposited in the deep lung was more than Case 1. Although the current model is not capable of returning aerosols that reach the deep lung region to the 3D airway model during exhalation, this increased in upper bound predictions of deep lung deposition is consistent with previous studies that have suggested smaller particles to have been associated with increased hazard of inhalation anthrax in multiple animal models compared to larger particle sizes (Druett et al., 1953). Other major factors that profoundly affected spore deposition in the human model included the near 90° turn between the nasal and pharyngeal regions of the human that increases spore impaction and the laryngeal anatomy that leads to secondary currents and more abrupt changes in airflow directions than occurs in the rabbit. In the rabbit model, on average 56.64% (Case 1 and Case 2) of the total exposed spores escaped the 3D outlets, compared to 62.08% escaping the 3D outlets in the human model. During the exhalation phase of the breathing cycle, an average of 28.18% of the total inhaled spores exited the rabbit nostril, whereas 28.04% exited the human nostril during the exhalation phase. While 3D CFD aerosol simulations provides important insights into species- and site-specific deposition patterns of inhaled spores that could be important to the risk of disease, there are several limitations to the current approach that should be highlighted. The first of which was already addressed–the assumption that the spores that exit the 3D domain during the inhalation phase of the breathing cycle represents an upper bound on the amount of spores deposited in the distal lung. This assumption is necessary and reflects current limitations of Computational Fluid and Particle dynamics (CFPD) model of the respiratory system. Based on experimental data from Heyder, Armbruster, Gebhart, Grein, and Stahlhofen (1975) and Raabe et al. (1988), both rabbit and the human models overestimate distal lung deposition. As a result, we are currently working on methods for two-way communication between airway outlets in 3D CFD particle tracking models and lower dimensional particle tracking models for the deep lung similar to the two-way 3D-1D airflow models we’ve previously developed (Kuprat et al., 2013) that will ultimately improve predictions over the complete breathing cycle. The spores were also assumed to be non-interacting, an assumption that is reasonable for the low exposure concentrations used in the current study. However, at high concentrations, agglomeration of spores could occur which would increase upper airway deposition due to inertial impaction (Anjilvel & Asgharian, 1995). Three other limitations due to way the current state-of-the-art 3D/CFD models address respiratory physiology also affects predicted aerosol deposition across species. First, the application of non-physiological zero-pressure boundary condition at the 3D outlets, while justifiable due to similar terminal airway diameters and lack of data, does not address the heterogeneities in tissue mechanics in airways distal to each 3D airway outlet that could alter airflow profiles and thus, deposition efficiencies in each airway region. Second, the use of rigid airways that ignore pulmonary airway compliance as well as movement of larynx during respiration could also affect regional spore deposition. Third, the rabbit lung was cast at TLC in situ thereby preserving the lung morphology and maximizing the number of airways captured during the μCT imaging. However, differences in morphology are expected between the current model geometry and rabbit breathing at tidal volume. By not accounting for moving airways, the current model is expected to over predict deposition (Ryan, King, Larcombe, & Mullins, 2013). A number of researcher groups, including our own, are working on these limitations to account for heterogeneities in airway and tissue mechanics over the breathing cycle in animal and human models as well as the impact of underlying pulmonary disease on aerosol deposition (Darquenne, Lamm, Fine, Corley, & Glenny, 2016). Finally, with regard to B. anthracis spore deposition, the current model ignores the impact of mucociliary transport and host immune cell clearance mechanisms. Thus, all current predictions reflect initial deposition patterns only. We are currently working on species differences in clearance, cell uptake, germination, and proliferation rates to update current biokinetic models that relate exposures to local doses and ultimately responses.

6. Conclusions This study represents the first fully transient simulation of B. anthracis spore deposition in anatomically correct, expanded airway models of the New Zealand White rabbit and human. These models provide realistic, time-dependent, species- and site-specific deposition of spores in the rabbit and human respiratory geometry. Furthermore, these models can be used to refine local dose estimates that are incorporated into disease response models, regardless of whether the mode of action is via the jailbreak or Trojan horse mechanism. Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i

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Disclaimer The U.S. Environmental Protection Agency through its Office of Research and Development under Contract no. EP-C-09006 and Interagency Agreement DW9792343401 and the Department of Homeland Security, Science and Technology Directorate through Contract HSHQPM-14-X-00037 funded and managed the research described here for the development of the rabbit model and for spore simulations in both species. It has been subjected to the EPA and DHS review and has been approved for publication. Note that approval does not signify that the contents necessarily reflect the views of EPA and DHS. Mention of trade names, products, or services does not convey official EPA/DHS approval, endorsement, or recommendation. Development of the human model was performed under a Grant from the National Heart, Lung, and Blood Institute of the National Institutes of Health (R01 HL073598).

Acknowledgments The authors would like to thank Drs. Eletha Brady-Roberts, John Lipscomb and Jacky Rosati of the U.S. EPA for their valuable comments and feedback. A portion of this research was performed using PNNL Institutional Computing at Pacific Northwest National Laboratory.

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Please cite this article as: Kabilan, S., et al. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways. Journal of Aerosol Science (2016), http://dx.doi.org/10.1016/j.jaerosci.2016.01.011i