# Positron emission tomography-based comparison of methods for exposing macaques to respiratory pathogens

Benoît Delache, Anaïs-Rachel Garnier, Cécile Herate, Francis Relouzat, Pierre Lê-Bury, Julien Lemaitre, Asma Berriche, Quentin Sconosciuti, Eleana Navarre, Noémie Verguet, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Benoît Delache, Anaïs-Rachel Garnier, Cécile Herate, Francis Relouzat, Pierre Lê-Bury, et al. "Positron emission tomography-based comparison of methods for exposing macaques to respiratory pathogens." Journal of Aerosol Science, 2025, 189, pp.106646. 10.1016/j.jaerosci.2025.106646. hal-05150776

HAL Id: hal-05150776

https://hal.science/hal-05150776v1

Submitted on 8 Jan 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

# Positron emission tomography-based comparison of methods for exposing macaques to respiratory pathogens

Benoît Delache<sup>a,*</sup>, Anaïs-Rachel Garnier<sup>a</sup>, Cécile Herate<sup>a</sup>, Francis Relouzat<sup>a</sup>, Pierre Lé-Bury<sup>a,b,c</sup>, Julien Lemaitre<sup>a</sup>, Asma Berriche<sup>a</sup>, Quentin Sconosciuti<sup>a</sup>, Eleana Navarre<sup>a</sup>, Noémie Verguet<sup>d</sup>, Justina Creppy<sup>a,e</sup>, Olivier Gorgé<sup>d</sup>, Jean-Nicolas Tournier<sup>d</sup>, Frédéric Ducancel<sup>a</sup>, Laurent Vecellio<sup>e</sup>, Roger Le Grand<sup>a</sup>, Thibaut Naninck<sup>a,**</sup>

## ARTICLE INFO

Handling Editor: Chris Hogan

Keywords:

Respiratory tract deposition

Aerobiology

Non-human primate model of infectious
disease

PET/CT

## ABSTRACT

Non-human primates (NHPs) are relevant models for studies of human respiratory infections due to their similar anatomy and susceptibility to human pathogens, resulting in comparable disease manifestations following exposure via aerosols or liquid instillation. An understanding of pathogen deposition in the respiratory tract (RT) of NHPs according to the method of exposure is essential for infectious disease modeling. Here, we evaluated and compared three conventional exposure systems commonly used to replicate human RT infections: liquid endotracheal instillation (IT), facemask (FM) aerosol inhalation, and head-only exposure (HOE) aerosol inhalation. Using PET/CT imaging with [¹⁸F] fluorodeoxyglucose ([¹⁸F]FDG) as the radiotracer, we quantified deposition across the upper respiratory tract (URT), lower respiratory tract (LRT), and digestive tract in anesthetized, spontaneously breathing cynomolgus macaques. A 98.0 ± 1.4 % deposited dose in the LRT was obtained with IT, whereas FM gave only 28.2 ± 6.4 % (MMAD: 3.1 µm GSD 2.2) and HOE gave 40.4 ± 19.0 % (MMAD: 1.9 µm GSD 2.0). This variability of deposition rates highlights the need for precise metrology tools. The homogeneity of lung deposition was improved and ratio between peripheral deposition/central deposition (P/C ratio) were higher with FM, and particularly with the HOE device, than with IT. An in vivo study of macaques inhaling *Bacillus atrophaeus* spore suspensions tracked with [¹⁸F]FDG revealed a correlation between radioactivity and spore concentration in respiratory samples (nasal/tracheal swabs, bronchoalveolar lavage) after inhalation. In conclusion, exposure systems significantly affect pathogen dose deposition and distribution within NHP airways. Methods should thus mimic inhalation scenarios to improve vaccine or countermeasure efficacy trials against natural or intentional biological threats exposure. Finally, PET/CT imaging provides a robust tool for

<footnote><sup>a</sup> Université Paris-Saclay, Inserm, CEA, Center for Immunology of Viral, Auto-immune, Hematological and Bacterial Diseases (IMVA-HB/IDMIT/UMRS1184), Fontenay-aux-Roses & Le Kremlin-Bicêtre, France</footnote>

<footnote><sup>b</sup> Institut Pasteur, Université Paris Cité, Yersinia Research Unit, F-75015, Paris, France</footnote>

<footnote><sup>c</sup> Institut Pasteur, Université Paris Cité, WHO Collaborating Research and Reference Centre for Plague FRA-146, F-75015, Paris, France</footnote>

<footnote><sup>d</sup> Bacteriology Unit, Microbiology and Infectious Diseases Department, Institut de Recherche Biomédicale des Armées, 91220, Brétigny-sur-Orge, France, CNR-LE Charbon</footnote>

<footnote><sup>e</sup> Université de Tours, Centre d'Étude Des Pathologies Respiratoires, INSERM U1100, Tours, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: benoit.delache@cea.fr (B. Delache), thibaut.naninck@cea.fr (T. Naninck).</footnote>
monitoring and controlling exposure, decreasing the number of animals required for studies through precise dose control and tissue targeting.

# 1. Introduction

The transmission of respiratory infections poses a significant public health concern worldwide, as demonstrated by the recurrent outbreaks caused by influenza viruses, coronaviruses, respiratory syncytial virus or *Mycobacterium tuberculosis*. These microbes, characterized by their ability to spread through respiratory droplets, aerosols or direct fomite contact, place healthcare systems under pressure and cause substantial morbidity and mortality (Li, 2021). Tuberculosis, a strictly aerosol-transmitted infection, accounted for 1.25 million deaths worldwide in 2023 (Global tuberculosis report, 2024). The World Health Organization reports that lower respiratory tract (LRT) infections remain the most deadly type of contagious disease in the world, ranking fourth among the leading causes of death (https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death). Multiple factors, including biological and physical mechanisms, underlie the complexity of respiratory pathogen transmission (Leung, 2021). Depending on the mechanism of particle formation, the size of the particles containing the infectious agents can vary in size, ranging from droplets (10–300 µm) subject to ballistic effects, to smaller aerosolized particles (0.1–20 µm) (Zhou & Zou, 2021). There is a consensus that there is a risk of airborne transmission when the pathogen is present and replicative within a range of respiratory droplet sizes extending from 100 nm to 100 µm (Li et al., 2021; Wang et al., 2021a). Furthermore, the presence of influenza virus, SARS-CoV-2 or *Mycobacterium tuberculosis* in exhaled particles produced by infected individuals has been demonstrated. The particles have a polydisperse size distribution that is greatly influenced by the mechanism of droplet production (Madas et al., 2020; Zhou & Zou, 2021). Coughing, in particular, leads to the production of pathogen-containing particles of 1 µm–10 µm in size, whereas breathing produces smaller particles, in the range of the respirable fraction of aerosols (<5 µm) (Fennelly, 2020; Yan et al., 2018). Airborne infection with inhaled particles is thought to occur at particle sizes below 10 µm, with the deposition of 5–10 µm particles mostly in the upper respiratory tract (nose and throat) and the inhalation of particles of less than 5 µm in size (respirable fraction) targeting the bronchi (5–2.5 µm) and the alveolar space (<2.5 µm) (Roy et al., 2010) in humans. The way in which an individual is exposed to a respiratory virus or bacteria can have a major impact on the host response to infection and pathophysiology (Bixler et al., 2022; Hofer et al., 2021; Marriott et al., 2016; Mooij et al., 2021a). A similar situation has already been demonstrated in macaque and hamster COVID-19 models (Bixler et al., 2022; Boydston et al., 2023). In the same manner, the dose and method of administration of the influenza virus affect the development of the host immune response and the onset of symptoms in both humans and macaques (Memoli et al., 2016; Mooij et al., 2021a). In addition, the influence of inhaled infectious particles size on disease severity has also been demonstrated in bacterial infection models (Bartrand et al., 2008; Day & Berendt, 1972; Druett et al., 1956). For explorations of respiratory infectious diseases, an ideal animal model should reproduce the human route of infection and associated symptoms as closely as possible (Phalen et al., 2008). Due to their anatomic, phylogenetic, and immune system similarities to humans, non-human primates (NHP), and macaques in particular, are particularly relevant for vaccines and therapeutic efficacy preclinical trials (Gardner & Luciw, 2008; Lemaitre et al., 2021). However, several methods are used to administer respiratory pathogens to macaques in such studies. The intratracheal instillation of a liquid inoculum, often coupled with intranasal deposition, is the one of most common method used as well as the aerosol route. In literature, the aerosol methods are used, with a facemask or head-only exposure (HOE) devices. Published findings highlight the major influence of the method of administration on the distribution of aerosolized pathogens in the RT and, consequently, the induction of the pathophysiological response (Creppy et al., 2024). However, the distribution of the infectious inoculum in the respiratory tract according to the method of pathogen administration has not been documented for cynomolgus macaque (CM) infectious disease models. It is therefore difficult to interpret the exposure scenarios reproduced during challenges. Furthermore, nuclear imaging is traditionally used in aerosol therapy development to determine the aerosol deposition area and doses of medication inhaled, but it has not been systematically used in aero-biology research and animal model development (Dolovich, 2001; Mairinger et al., 2022). One previous study used a gamma camera to compare the influence of administration methods for a therapeutic aerosol in macaque lungs (Beck et al., 2002). However, this study did not compare HOE with other methods and used a limited two-dimensional analysis of deposition in airways. Moreover, PET/CT provides a better three-dimensional (3D) visualization of the anatomy of the subject and radiotracer distribution than scintigraphy (Dolovich & Bailey, 2012; Fleming et al., 1996). This technology has been used for a number of years as a replacement for gamma cameras, with [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) in particular, to trace therapeutic molecules in the upper and lower airways (Dolovich, 2001; Lee & Berridge, 2002). [¹⁸F]FDG inhalation has already been used as a non-invasive method for describing the anatomical areas targeted as a function of monodispersed aerosol particle size in macaques and its feasibility and the importance of conducting metrology preclinical studies in vivo have been demonstrated (Dabisch et al., 2017). The primary objective of this study was to use PET/CT to determine the distribution of inoculum in macaque airways following pathogen administration by three different methods: intratracheal liquid instillation (IT), aerosol inhalation by nebulization with a facemask (FM) and aerosol inhalation by nebulization inside a head-only exposure (HOE) device. The secondary objective was to evaluate the utility of [¹⁸F]FDG for imaging and quantifying pathogen aerosol deposition in CM airways by PET/CT. *Bacillus atrophaeus* spores were used as a model pathogen for inhalation selected because of the relatively large size of spores which can cause difficulties in plugging mesh orifices, their ability to survive in hostile environment for a long time and the relative ease of use due to their low pathogenicity.
## 2. Materials and methods

### 2.1. Ethics statement

Cynomolgus macaques (Macaca fascicularis), aged 4–9 years, with a body weight of 5.07 ± 0.7 kg (10 females, 8 males), originating from Mauritian AAALAC certified breeding centers were used in this study (weight distribution by group detailed in Table 1). All animals were housed in the IDMIT infrastructure facilities (CEA, Fontenay-aux-Roses) under BSL-3 containment (animal facility authorization D92-032-02, Prefecture des Hauts de Seine, France) and in compliance with European Directive 2010/63/EU, French regulations, and the Standards for Human Care and Use of Laboratory Animals of the Office for Laboratory Animal Welfare (OLAW, assurance number A5826-01, United States). The protocols were approved by the institutional ethics committee “Comité d’Ethique en Expérimentation Animale du Commissariat à l’Energie Atomique et aux Energies Alternatives” (CEtEA n° 44) under statement number A20_066. The study was authorized by the French Ministry of Research, Innovation and Education under registration number (APAFIS#29191–2021011811505374 v1).

### 2.2. Macaque respiratory tract exposure methods

Animals were anesthetized with ketamine (Imalgene 1000, 10 mg/kg, intramuscular route) and medetomidine (Domitor, 10 µg/kg, intramuscular route) and were exposed in a class II biological safety cabinet. The animals were placed in the dorsal decubitus position on a specific restraint cushion throughout the exposure procedure. [¹⁸F]FDG was delivered alone by IT, FM and HOE to compare the distribution of radioactivity achieved with these three methods in the airways of six macaques for each method. A mixture of [¹⁸F]FDG and *Bacillus atrophaeus* spores was nebulized via a FM to evaluate the utility of [¹⁸F]FDG for quantifying *Bacillus atrophaeus* spore deposition in three macaques.

#### 2.2.1. Intratracheal liquid instillation

A laryngoscope was used to visualize the epiglottis and larynx (Fig. 1a). A mark was made on a nasogastric tube (2.6 mm diameter/ 40 cm COVETO ref: 706427) introduced into an endotracheal tube (3 mm diameter/16 cm COVETO ref: 706188) to ensure that the lower extremity of the nasogastric tube was placed 1 cm below the lower extremity of the endotracheal tube. The animals were intubated with the endotracheal tube, with the balloon placed just below arytenoid cartilages. The radioactive preparation contained 41 ± 0.4 MBq [¹⁸F]FDG in a total volume of 1 mL/kg. The nasogastric tube was introduced into the endotracheal tube, stopping at the mark; a 10 mL syringe was connected to the tube and [¹⁸F]FDG was injected into the trachea without flushing the dead volume within the nasogastric tube, to limit the risk of radioactive contamination during syringe handling. Ten minutes after instillation, a CT scan was performed and positron emission data were acquired, to ensure that the same time elapsed before imaging regardless of the exposure method.

#### 2.2.2. Aerosol inhalation with a facemask or head only exposure device

The aerosol was administered during spontaneous breathing without oxygen supplementation (Fig. 1b & c). Oximetry was performed and temperature, respiratory and heart rates were monitored during administration, until complete recovery. The same vibrating mesh nebulizers (Aeroneb® Solo, Aerogen, Galway, Ireland) were used with the FM and HOE devices. A mesh nebulizer was chosen because such nebulizers maintain the viability of fragile respiratory viruses more effectively than jet nebulizers (Kanekiyo et al., 2023; Mooij et al., 2021b; Wonderlich et al., 2017). In both configurations (FM and HOE), an electric generator was used to control the vibration frequency of the mesh (Emka Technologies®); it was set at 10% of the maximum frequency to nebulize the total volume in 10 ± 2 min, corresponding to an inoculum flow of about 100 µL/min.

For facemask (FM) exposure (Fig. 1b), a specific airtight facemask (Intersurgical, 15M, size 1) connected by Y and T piece tubing and including one-way low-resistance valves was used to administer the aerosol generated by the Aeroneb® Solo vibrating mesh

<caption>**Table 1**
Experimental conditions and summary of deposition performances.</caption>

Description of the animals used for the *in vivo* study, including weights and sample sizes. The characteristics of the aerosol generated (MMAD ± SD and GSD ± SD) within the FM or HOE are shown, together with the deposition performance (means ± SD) in terms of total dose deposited and the distribution of the deposited dose between the digestive tract (stomach and esophagus), upper respiratory tract (URT) and lower respiratory tract (LRT).

<table><thead><tr><th>Administration</th><th>Animals</th><th colspan="2">Aerosol metrology</th><th>Total deposition/ inoculum loaded</th><th>Digestive/ deposited</th><th>URT/ deposited</th><th>LRT/ deposited</th></tr></thead><tbody><tr><td>Methods</td><td>Number</td><td>Weights ± SD</td><td>MMAD ± SD</td><td>GSD ± SD</td><td>Means ± SD</td><td>Means ± SD</td><td>Means ± SD</td></tr><tr><td>Liquid I.T. Instillation</td><td>n = 6</td><td>5.1 ± 0.8 kg</td><td>NA</td><td>NA</td><td>81.4 ± 2.5 %</td><td>0.1 ± 3.9 %</td><td>0.1 ± 0.1 %</td></tr><tr><td>Aerosol Facemask</td><td>n = 6</td><td>4.8 ± 0.8 kg</td><td>3.1 ± 0.2 μm</td><td>2.2 ± 0.3</td><td>32.2 ± 6.6 %</td><td>28.9 ± 9.5 %</td><td>42.8 ± 9.3 %</td></tr><tr><td>Aerosol HOE</td><td>n = 6</td><td>5.3 ± 0.7 kg</td><td>1.9 ± 0.1 μm</td><td>2.3 ± 0.1</td><td>9.8 ± 4.2 %</td><td>31.5 ± 8.3 %</td><td>28.1 ± 10.7 %</td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Respiratory tract administration methods adapted for macaques</figcaption></figure>

Schematic representation of the three respiratory tract exposure methods. Anesthetized animals were exposed to [¹⁸F]FDG while spontaneously breathing and in a dorsal position.

a) Intratracheal liquid instillation (IT). After intratracheal intubation, a nasogastric tube was inserted into the intratracheal tube for inoculum administration. An inoculum of 1 mg/kg (40 MBq [¹⁸F]FDG) was injected into the tube with a syringe.

b) Aeroneb® facemask (FM) exposure. Commercial Aeroneb® Solo vibrating mesh nebulizers were adapted to Y-form tubing circuits with air filtration. A pediatric resuscitation facemask was fixed in place over the face of the macaque with straps. The direction of flow of the aerosol was controlled with low-resistance valves positioned upstream (inhalation) and downstream (exhalation). An inoculum of 1 mg/kg (40 MBq [¹⁸F]FDG) was nebulized over 10 min with an EMKA mesh frequency controller (10 % max flow corresponding to ±100 µL/min).

c) Aeroneb® head-only exposure. Aeroneb® Solo vibrating mesh nebulizers were adapted to fit a macaque head-only exposure device (total volume 16 L). Dry airflow (6 L/min) transported the aerosol generated by the Aeroneb® mesh (mesh frequency at 10 % max flow corresponding to ±100 µL/min) into the inhalation chamber for the animal to inhale. The aerosol concentration was measured after aspiration (1 L/min), with two gelatin filters connected to the chamber in series. The radioactivity trapped in the filters was divided by the duration of exposure to obtain the concentration of [¹⁸F]FDG/liter of aerosol for dose calculation using macaque's $V_{min}$.

nebulizer. The reservoir was loaded with 1 mL of a solution containing 36 ± 8 MBq [¹⁸F]FDG, which was nebulized at a rate of 100 µL/min (duration 10 min ± 2 min) for the comparison with IT and HOE. *Bacillus atrophaeus* spores in a total volume of 1 mL (0.5 mL of 2.0x10⁸ colony-forming units (CFU)/mL in 0.9 % NaCl+0.01 % Tween 20 mixed with 0.5 mL of 0.9 % NaCl containing 40 MBq [¹⁸F]FDG) were loaded into the reservoir and nebulized at a rate of 100 µL/min (duration 10 min ± 2 min) for FM inhalation and spore deposition characterization in macaque airways.

For HOE exposure (Fig. 1c), a T piece (Aerogen, Galway, Ireland) was connected to a dedicated head-only exposure system (CH Technologies®, Westwood, New Jersey, USA) for macaque pathogen exposure. We passed 6 L/min fresh dry filtered air through the HOE with a medical compressor connected to the Aerogen T piece just ahead of the HOE. The percent humidity (RH%) inside the HOE was measured before and immediately after nebulization (TH110 Sauermann®, Montpon-Ménestérol France). The RH% was 25 ± 1 % at the start and 58 ± 2 % at the end of nebulization. Aerosol particle concentration was measured in real time with a PALAS® optical counter, with collection of the aerosol during each exposure with two gelatin filters (presterilized gelatin filter, 25 mm SKC®, Dorset, UK) mounted in series on a suction channel set at 1 L/min for aerosol concentration measurement by gamma counting. Once the 10-min nebulization was complete, the aerosol concentration in the chamber decayed asymptotically. The animals' heads were taken out 3 min after nebulization ceased, and aspiration on the filter was stopped concurrently. Details regarding the parameters and calculations for aerosol generation are available (Supplementary material).

## 2.3. Particle size distribution determination with a cascade impactor and aerosol sampling with an impinger

We characterized the particle size distribution of the [¹⁸F]FDG nebulized alone or mixed (40 MBq/1 mL) with a suspension of *Bacillus atrophaeus* spores (10⁸ CFU/mL). The *Bacillus atrophaeus* spore suspension was prepared at a final concentration of 1.0x10⁸ CFU/mL in 0.9 % NaCl+0.01 % Tween 20. Particle size distribution was characterized by cascade impaction with a Dekati® Low Pressure Impactor (DLPI+) operating at 40 Mbar and ambient temperature (22 ± 2 °C). Nebulization was performed over a period of 3 min to prevent plate saturation, corresponding to an estimated volume of 300 µL. After nebulization, each plate of the impactor was rinsed with 4 mL 0.9 % NaCl. The radioactivity (Bq) and the number of spores (CFU) were quantified and the mass distribution was calculated for each plate. [¹⁸F]FDG activity was measured by gamma counting (Hidex AMG, LabLogic). Culture was performed on brain heart infusion (BHI) agar after multiple dilutions to maximize accuracy and colonies were counted after 24 and 48 h of culture at 37 °C. Data are expressed as the normalized distribution of the total mass of radioactivity deposited on all stages of the impactor and the mean ± standard deviation (SD) calculated (n = 3 runs for each interface: facemask or HOE device and for each liquid preparation: [¹⁸F]FDG alone and with *Bacillus atrophaeus* spores). The *Bacillus atrophaeus* spore suspension (1.0x10⁸ CFU/mL in 0.9 % NaCl+0.01 % Tween 20) was nebulized with the same Aeroneb® nebulizer, using a T piece, and was collected in a glass impinger containing 1 × 0.9 % NaCl at an airflow rate of 12.5 L/min (biosampler, SKC®, Dorset, UK). The number of CFU was determined before nebulization and
after nebulization and sampling (biosampler) to evaluate the effect of nebulization on the viability of *Bacillus atrophaeus*.

## 2.4. PET/CT imaging and deposition analysis

We characterized deposition patterns by measuring [¹⁸F]FDG activity in the digestive, upper and lower respiratory tracts of macaques for the three selected exposure methods (IT versus FM versus HOE). The 3D resolution of the PET/CT scan also provided the information required to determine the lobar distribution and pulmonary deposition in central and peripheral lung. We used positron emission tomography coupled to computed tomography (PET/CT) imaging (Vereos PET/CT, Philips Healthcare) implemented in animal biosafety level 3 (ABSL-3) facility. Macaques were exposed to 1 mL/kg inoculum for instillation or 1 mL of inoculum for [¹⁸F]FDG (41 ± 0.4 MBq for IT or FM and 109 MBq ± 26 MBq for HOE) aerosol exposure via mesh nebulization (Aeroneb®) as described above. The CT scan was initiated immediately after the end of exposure (about 10 min after [¹⁸F]FDG instillation or just after nebulization) and macaques were scanned from the base of the skull to the knees, including the lungs and abdominal cavity. We used a CT detector collimation of 64 × 0.6 mm, a tube voltage of 120 kV and an intensity of about 130 mA. Whole-body CT images were reconstructed with a slice thickness of 1.5 mm and a pixel spacing of 0.25. A whole-body PET scan (5 bed positions, 3 min/bed position) was performed over a period of about 20 min after exposure. The PET images were reconstructed on a 256x256 matrix (3 iterations, 17 subsets).

PET/CT image analysis was performed with 3DSlicer (open-source tool) and LIFEx version 6.00 software (Nioche et al., 2018). For segmentation, various regions of interest were contoured semi-automatically on the basis of anatomical information. A 3D volume of interest (VOI) was interpolated from several regions of interest (ROI) in different image slices, to cover the entire organ or anatomical structure. The radioactive signal in each VOI was determined and expressed in Becquerels (Bq). The distribution of the inoculum within the body was followed in different compartments: i) the upper respiratory tract (URT), from the entrance of the nose to the vocal cords, ii) the digestive tract, from the beginning of esophagus (ahead of the vocal cords) to the rectum and iii) the lower respiratory tract (LRT), corresponding to the thoracic respiratory tract (whole lungs and trachea). We used the LRT VOI to assess the lobar distribution of the inoculum by segmenting the lobes within this VOI. The lobes were defined by following the main fissures in the lung, the right horizontal and oblique fissures, the left oblique fissure and the left mediocranial fissure. We were able to identify seven lobes — cranial right, middle right, caudal right, cranial left lingula, cranial left culmen, caudal left and accessory — in the LRT VOI. According to normalized anatomical segmentation, we assumed that each of these lobes accounted for one seventh of the total volume of the lung. The volumetric percentage of each lobe was therefore 14.3 % (100:7).

We compared inoculum penetration between the three methods through a scintigraphic assessment of krypton (Kr) gas deposition. For scintigraphy, a rectangular ROI was defined for each lung at the boundaries of the krypton ventilation scan (15 % of the peak Kr counts). This rectangle was then split into two parts — a central ROI and a peripheral ROI — with one quarter of the area occupied by the central ROI and three quarters occupied by the peripheral ROI. The dimensions of the central ROI were defined as a width half that of the whole-lung ROI and a height one half that of the whole-lung ROI, centered on the middle of the interior boundary. The peripheral ROI was defined as the rest of the rectangular ROI (Brand et al., 2009). The use of PET/CT imaging techniques made it possible to determine the rectangular ROI according to the actual anatomical boundaries of the lung in the frontal and sagittal planes of the CT scan. We used the same delimitation for the central ROI as on scintigraphy (half of the width and half the height of the whole-lung ROI). Individual specific features on segmentation were adapted according to the second bronchial division rather than the middle of the interior boundary. By using the second division as a landmark, we were able to ensure that most of the proximal conductive airways were within the central VOI. As for scintigraphy, the rest of the VOI was considered to be the peripheral VOI. We then used the 3D properties of the CT scan to add another delimitation, this time in the transverse plane of the CT scan, dividing the central ROI in two. This made it possible to retain only the dorsal half of the central VOI. This 3D segmentation technique resulted in a central VOI to peripheral VOI ratio of 1:3. For all lobes and all animals, we calculated the percent volume of each compartment (peripheral and central) after anatomical CT analysis to confirm that the positioning for segmentation was correct. The percentages obtained were 75 ± 7 % for the peripheral VOI and 25 ± 7 % for the central VOI, providing an indicator of quality control for volumetric positioning for each individual. A uniform volumetric deposition for penetration would yield a ratio between peripheral deposition/central deposition (P/C ratio) of one. Finally, a lower index is associated with more central deposition.

## 2.5. Inhalation of radioactive particles containing spores in macaques

Three CM were exposed to a suspension of spores (1.0x10⁸ CFU/mL in 0.9 % NaCl+0.01 % Tween 20) containing 40 MBq [¹⁸F]FDG in a total volume of 1 mL over a duration of 10 ± 2 min via a facemask with the Aeroneb® nebulization device. A thoracic CT scan was performed immediately after inhalation. Positron emission was then acquired over a period of 20 min, to visualize and quantify the doses of the spore and radiotracer mixture deposited in each compartment, as described above. Once imaging was completed (30 min after the start of nebulization), we collected respiratory fluid samples from each of the three macaques. Nasopharyngeal swabs from each nostril, tracheal swabs, and bronchoalveolar lavage (BAL) were taken (duration of sampling 10 min). The samples were passed through a gamma counter for radioactivity quantification and then stored at +4 °C for 24 h to allow decay before culture for CFU quantification. CFU counts were obtained (number of colonies determined after 24 h and 48 h of culture on BHI agar as described above) and compared with the calculated CFU after radioactivity quantification. The spores have been shown to survive in liquid media for extended periods, making it possible to wait for radioactive decay to occur (24 h for [¹⁸F]FDG) before setting up cultures to determine the number of CFU in samples.
## 2.6. Statistical analysis and figures

GraphPad Prism 8 and R v.4.3.3 were used to create all the figures and to perform the statistical analysis. Nonparametric tests were performed because the data were non-normally distributed and the number of animals was small. Weighted linear regression was done using the lm function from the "stats" base package in R v4.3.3, with a 1/x² weighting to account for greater variance of the higher values. Biorender was used to generate Figs. 1 and 6a.

# 3. Results

## 3.1. Aerosol characterization

For comparison of the methods of aerosol inhalation (FM and HOE), we first characterized particle size with a cascade impactor (Fig. 2). The mass median aerodynamic diameter (MMAD) of the aerosol produced was measured in triplicate for both aerosol exposure method, and the SD of the triplicates was determined. The geometric standard deviations (GSD) were calculated from the particle size mass distributions obtained, and the SD of the triplicates was determined. The results for FM and HOE were MMAD 3.1 ± 0.2 µm GSD 2.2 ± 0.3 and MMAD 1.9 ± 0.1 µm GSD 2.3 ± 0.1, respectively, revealing two different polydisperse particle size distributions (Table 1) with significatively different descriptive statistics (Welch's unequal variance t-test, MMAD p = 0.0029; GSD p = 0.63). We observe a difference between the two particle size distributions, with virtually all particles smaller than 5.4 µm with HOE but not with FM (Fig. 2). This first result allows us to anticipate a potential difference in the distribution of aerosol deposits after inhalation of the aerosol.

## 3.2. The doses deposited and their distribution in macaque airways depend on the exposure method used

In order to compare inoculum deposition regarding methods selected, we exposed CM to [¹⁸F]FDG. Three dimension images were recorded after administration of [¹⁸F]FDG, for each of the three exposure techniques: Liquid instillation IT (Fig. 3a), aerosol Facemask FM (Fig. 3b) and aerosol Head Only Exposure HOE (Fig. 3c), Supplementary videos. Three cross-sections were used to assess [¹⁸F]FDG distribution: a maximum intensity frontal projection (right images), a transverse section (upper left), and a sagittal section (lower left). For liquid intratracheal instillation, we observed a predominantly dorsal deposition (visible in the transverse section) with a non-homogeneous lobar distribution, primarily concentrated on the right side (frontal section). The non-negligible residual radioactivity within the nasogastric tube is visible on the frontal image. Regarding inhalation methods, the second panel shows deposition of the [¹⁸F]FDG inhaled by FM and its distribution throughout the respiratory tract, with high levels of deposition in the head and more

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Aerosol metrology measured with a cascade impactor for the FM and HOE methods</figcaption></figure>

The [¹⁸F]FDG mass distribution over aerosol particle sizes generated by the Aeroneb® Solo vibrating mesh nebulizer was normalized as a percentage of total deposition. Mean radioactivity mass deposition percentage is shown with the standard deviation (n = 3 nebulizers for FM and n = 3 for HOE) on the 14 plates of a DLPI+ cascade impactor, as a histogram. Results facemask (FM) are shown in light blue, and those for head-only exposure (HOE) are shown in blue aqua. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
a

Liquid Instillation (IT)

<figure><img src="image_7.png" /></figure>

b

Aerosol Facemask (FM)

<figure><img src="image_8.png" /></figure>

c

Aerosol Head Only Exposure (HOE)

<figure><img src="image_9.png" /></figure>

d

% Inoculum deposited

<figure><img src="image_10.png" /></figure>

e

% Digestive/Deposited

<figure><img src="image_11.png" /></figure>

f

% URT/Deposited

<figure><img src="image_12.png" /></figure>

g

% LRT/Deposited

<figure><img src="image_13.png" /></figure>

(caption on next page)
<caption>**Fig. 3.** Distribution of [¹⁸F]FDG after respiratory tract administration in CM.</caption>

a, b, c) Representative CT scan images and positron emission of [¹⁸F]FDG deposited after administration by the three methods tested. From left to right: a) Liquid intratracheal instillation (IT), b) Aeroneb® facemask inhalation (FM) and c) Aeroneb® head-only exposure inhalation (HOE). For each image, transverse sections are shown in the top left corners, sagittal sections are shown in the bottom left corners and whole-body frontal sections are shown on the right. A semi-quantitative scale for standardized uptake values (SUV) was used to visualize the deposition gradients (Blue < SUV < Red).

d, e, f, g) Distribution of [¹⁸F]FDG within the CM after administration by the three methods (IT in dark blue, FM in light blue and HOE in blue aqua). Individual exposure values are shown, with means and standard deviations. Black horizontal bars indicate the results of comparisons between the IT, FM and HOE groups in two-tailed Mann-Whitney tests with *p*-values below the bars (ns: not significant, \*: *p* < 0.05, \*: \*: *p* < 0.01). d) Percentage of the total inoculum deposited (digestive, URT and LRT) relative to the radioactivity dose loaded into the syringe or nebulizer. e,f,g) Distribution of total deposited [¹⁸F]FDG within RT compartments after 3D segmentation: e) Digestive tract (stomach and esophagus), f) URT (nostrils, throat, pharynx) and g) LRT (lungs). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

moderate deposition in the lungs. Radioactivity was detected on both sides of the lungs (frontal section), and in the upper respiratory tract and stomach (in red below the lungs). The most distal regions of the lobes appeared to display less deposition than the tracheobronchial areas, particularly on the frontal and transverse sections. Deposition was also observed on the faces of the animals, under the mask (sagittal section). Finally, [¹⁸F]FDG aerosol deposition following inhalation by HOE is visible in the third set of images. Deposition was observed predominantly in the nostrils, esophagus, and lungs. The transverse section revealed a relatively uniform distribution of radioactivity across the pulmonary area. The sagittal section shows non-negligible radioactivity deposition on the fur at the back of the heads of the animals.

The total dose deposited in the respiratory and digestive tracts relative to the initial dose loaded into the device (syringe or nebulizer) was calculated. The mean dose deposited (± SD) was 81.4 ± 2.5 % for IT, 32.2 ± 6.6 % for FM, and 9.8 ± 4.2 % for HOE after segmentation (Table 1).

Total deposition, regarding inoculum activity loaded, differed significantly, whatever the methods compared, and large losses were observed with aerosol methods, particularly for HOE in comparison with FM (*p*-value = 0.002) (Fig. 3d). To go further in the comparison, the distribution of 100 % of the deposited dose was then further detailed by digestive and respiratory tract region. We quantified the deposited dose inside three compartments of interest after 3D anatomical segmentation: the digestive tract (esophagus and stomach), the upper respiratory tract (nose, throat, pharynx), and the lower respiratory tract (trachea and lungs) (Fig. 3e, f, g, respectively). For IT liquid instillation, the deposited dose was found almost entirely in the lower respiratory tract (mean ± SD = 99.8 ± 0.2 %), because this method is not representative of inhalation exposure and does not involve the mechanisms of deposition in the upper airways. For aerosol inhalation routes, the deposited dose was found in the digestive tract, URT, and LRT. For FM, 28.9 ± 9.5 % was found in the digestive tract, 42.8 ± 9.3 % in the URT, and 28.2 ± 6.4 % in the LRT. For HOE, 31.5 ± 8.3 % was found in the digestive tract, 28.1 ± 10.7 % in the URT, and 40.4 ± 19.0 % in the LRT.

However, the distributions achieved with the two aerosol exposure methods were similar (FM versus HOE), although the particle sizes for these two methods were significantly different. This lack of significance for the distribution comparisons may be due to the small number of macaques studied (*p*-value = 0.7 for digestive, *p*-value = 0.14 for URT, and *p*-value = 0.48 for LRT).

We checked the administered doses by measuring [¹⁸F]FDG concentration with gelatin filters in the HOE during exposure, which made it possible to calculate the spray factor, corresponding to the radioactive dose per mL of aerosol divided by the radioactive dose per mL loaded into the nebulizer reservoir. The spray factor for [¹⁸F]FDG was highly reproducible, at 8.31E-6 ± 4.15E-7, under our experimental conditions (Supplementary Fig. 1). Furthermore, the dose presented ($C_{aero} \times V_{minute}$) was calculated from head-out plethysmography measurements taken just before inhalation of the aerosol or based on Guyton's formula. In our experimental conditions, the minute volume of the anesthetized animals was 801 ± 199 mL/min. We calculated the percentage of actual deposited radioactivity relative to the theoretical presented dose for each individual. Plethysmography data indicated that 115 ± 32.8 % of the inhaled dose was actually deposited in the HOE configuration, whereas 80 ± 43 % was deposited according to Guyton's formula: $V_{min} = 2.10 \times (body weight)^{0.75}$ showing variability between calculated and actual deposited dose (Supplementary Fig. 2).

### 3.3. Aerosol exposure is associated with a higher homogeneity of lobar deposition and higher levels of peripheral lung deposition than instillation

We explored the homogeneity of deposition across the seven pulmonary lobes in macaques, by normalizing the dose of radioactivity deposited in each lobe relative to the volume of the lobe concerned, as measured by CT (Fig. 4a). We used an analysis of variance to explore the homogeneity of lobar deposits for each administration method.

#### 3.3.1. Liquid IT instillation

As noted on qualitative visualization, instillation resulted in a statistically significant heterogeneity of deposition, mainly due to higher levels of deposition on the right side and in the caudal lobes (*p* < 0.0001). Conversely, the cranial and middle left lobes — referred to as the culmen and lingula, respectively — were much less targeted by instillation than the caudal lobes. The SD bars also revealed a high level of individual variability.
<figure><img src="image_14.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_15.png" /></figure>

(caption on next page)
<caption>**Fig. 4.** Lobar distribution and penetration of aerosol in the lung</caption>

a) Aerosol lobar deposition: [¹⁸F]FDG lobar distribution, by exposure method, after segmentation. The results obtained were normalized by lobe volume. The blue dotted line indicates lobe volume as a percentage relative to anatomical segmentation (100%/7 lobes = 14.3% per lobe). Values are shown for individual exposure (black dots), with means (histogram) and standard deviations (black lines). A schematic representation of the different lobes and their colour are shown to the right of the graph to facilitate reading. The four right lobes are shown in green (cranial Cr in bright green, middle M in mid-green, caudal Cd in dark green and accessory in sage green) and in pink the left lobes (culmen Cu in pale pink, Lingula Li in blush pink, caudal Cd in fuchsia). Analysis of the variance of lobar deposition based on Kruskal-Wallis tests with *p*-values shown below the bars. "ns" indicates an absence of significant difference between normalized lobar deposition values, whereas asterisks indicate significant differences, *: *p* < 0.05, **: *p* < 0.01, ***: *p* < 0.001.

b) Peripheral deposition/Central deposition ratio (P/C ratio): Histograms show the [¹⁸F]FDG inoculum distribution for the three methods for the right and left sides of the lung. A schematic representation of the central (red) and peripheral (blue) compartment is visible on the right of the graph to facilitate reading. The right side is shown in green, the left side in pink. A ratio of one, represented by the red dotted line on the graph, corresponds to the normal volumetric P/C ratio (3/4 peripheral and 1/4 central). Values shown are for individual exposure, with means and standard deviations. The black horizontal bar indicates the results of a comparison between lung sides (right versus left) or between methods, performed with a two-tailed Mann-Whitney test with *p*-values shown below the bars. "ns" indicates an absence of significant difference, whereas asterisks indicate significant differences, *: *p* < 0.05, **: *p* < 0.01, ***: *p* < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

### 3.3.2. Aerosol inhalation methods

FM gave a more homogeneous lobar deposition than liquid IT instillation, but statistical analysis nevertheless revealed an overall heterogeneity of deposition (p = 0.0015) driven by uneven deposition on the right side (p < 0.0001) despite homogeneity on the left side (p = 0.14). The right caudal lobe was preferentially targeted. Finally, HOE yielded a statistically less heterogeneous deposition across all seven pulmonary lobes (p = 0.08).

The ratio between peripheral deposition/central deposition (P/C ratio) was used to estimate the proportion of alveolar deposition relative to tracheal-bronchial deposition in animals (Fig. 4b). A schematic representation of this segmentation is shown on the right side of Fig. 4b. IT resulted in a P/C ratio of 0.3 ± 0.1 on the right side and 0.4 ± 0.3 on the left side; this difference was not statistically significant. The P/C ratio was 1.0 ± 0.4 on the right and 0.9 ± 0.2 on the left side of lung for FM, and 1.5 ± 0.7 on the right and 1.5 ± 0.5 on the left side of lung for the HOE. A statistical analysis of these data revealed no significant differences in P/C ratio between the right and left sides for any exposure method (p = 0.69 for IT, p > 0.99 for FM, and p > 0.99 for HOE). A comparison of P/C ratio between the three methods revealed a significant difference between IT and FM (p = 0.041), and between IT and the HOE (p = 0.009). However, there was no significant difference between the two aerosol inhalation methods probably due to the small number of individuals (p = 0.13).

<figure><img src="image_16.png" /><figcaption>**Fig. 5.** Validation of radioactive tracking by [¹⁸F]FDG of *Bacillus atrophaeus* nebulized (FM)</figcaption></figure>

Histograms represent distribution means and black lines standard deviation of percentage of mass aerodynamic diameter measured by cascade impactor after FM Aeroneb® nebulization (n = 3 nebulizers). One mL of 0.9% NaCl containing respectively 40 MBq of [¹⁸F]FDG or 10⁸ CFU of *Bacillus atrophaeus* spores were prepared than nebulized. The percentage of total mass deposition of the two different preparations shown: [¹⁸F]FDG in light blue and *Bacillus atrophaeus* spores in orange. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
## 3.4. [¹⁸F]FDG allows the tracking of inhaled *Bacillus atrophaeus* spores

### 3.4.1. In vitro evaluation of spore's tracking by [¹⁸F]FDG

We first assessed the impact of nebulization via the Aeroneb® on spore viability. Viable spore concentrations before and after nebulization did not differ significantly when analyzed with the all-glass impinger (p = 0.30 and n = 3). This result validates the use of the Aeroneb® mesh nebulizer for *Bacillus atrophaeus* spores, by demonstrating an absence of viability loss (Supplementary Fig. 3). We also compared the particle size distribution of *Bacillus atrophaeus* spores (CFU) to the particle size distribution of [¹⁸F]FDG radioactivity. These two particle size distributions were very similar, with no spores found in particles of less than 0.6 µm in size due to spore size (approximately 1 µm). Radioactive particles smaller than 0.6 µm were detected but accounted for less than 3.7 ± 0.7% of the total population (Fig. 5). The MMAD ± SD and GSD ± SD for [¹⁸F]FDG and *Bacillus atrophaeus* spores were calculated and compared (3.1 ± 0.2 µm and GSD 2.2 ± 0.3, and 3.8 ± 0.7 µm and GSD 2.1 ± 0.3, respectively). We found no significant difference between the two particle size distributions (Welch's unequal variance t-test, MMAD p = 0.27; GSD p = 0.7). These results therefore validate the use of [¹⁸F]FDG solution to track the deposition of *Bacillus atrophaeus* spores by an aerosol generated by an Aeroneb® mesh nebulizer.

### 3.4.2. In vivo validation of spore's tracking by [¹⁸F]FDG

CM (n = 3) were exposed by FM by inhalation of a suspension containing [¹⁸F]FDG (40 MBq) mixed with *Bacillus atrophaeus* spores (Fig. 6a). Nasopharyngeal swabs were collected from both nostrils (NS swabs left and right), together with tracheal swabs (T swabs) and total bronchoalveolar lavages (BAL). The radioactivity in the samples was measured with a gamma counter for proportional calculation of the predicted number of CFU for *Bacillus atrophaeus* spores using initial inoculum concentration. The predicted numbers of CFU were similar to the actual numbers of CFU counted in the same samples whatever the zone of deposition in the respiratory tract (Fig. 6b). We found no significant difference between the predicted and actual numbers of CFU for the 12 samples (two-tailed paired Wilcoxon test, p-value = 0.25), although only a small number of samples were considered. Weighted linear regression between predicted and measured CFU for the 12 respiratory tract samples showed a slope of 1.25 (p = 0.012, standard error SE = 0.41), highlighting a good linear relationship (Fig. 6c). Additionally, the regression intercept was of $10^4$ CFU (p = 0.19, SE = 7.10³). However, coefficient of determination R² was equal to 0.48, meaning that only half of the variability in the measures is explained by the difference in CFU doses, while the other half probably stem from experimental variability such as sampling methods or bacteria enumeration.

We concluded that radioactive tracing provided fairly reliable measurements of spore doses across different RT samples, in our experimental conditions, validating the feasibility of tracing *Bacillus atrophaeus* spore deposits by [¹⁸F]FDG PET detection. Taken together, these *in vitro* and *in vivo* results confirm that PET radioactivity measurements can be used for the quantitative assessment of *Bacillus atrophaeus* spore concentration along the respiratory tract 30 min after the end of exposure to a spore-containing aerosol by inhalation. Once the tracing had been validated, we performed a 3D analysis to estimate the doses of *Bacillus atrophaeus* spores deposited in the various compartments of the three individual CM. The total number of spores deposited in the RT and digestive tract was (mean ± SD) $3.1 \times 10^7 \pm 3.3 \times 10^6$. These spores were distributed as follows: $1.2 \times 10^7 \pm 2.5 \times 10^5$ for the digestive tract, $1.2 \times 10^7 \pm 8.8 \times 10^5$ for the URT, $2.0 \times 10^6 \pm 4.8 \times 10^5$ for the central lung and $5.3 \times 10^6 \pm 1.4 \times 10^6$ for the peripheral lung (Fig. 6d; Supplementary Table 1).

## 4. Discussion

Various methods are used to expose the respiratory tract of non-human primates to respiratory pathogens, with the aim of replicating natural transmission conditions as closely as possible (Miller et al., 2017). However, many studies relating to preclinical studies for infectious diseases indicate the total dose of pathogen presented without detailing its tissue distribution. Importantly, the RT composition (cells, exposed receptors), which may affect susceptibility to infection, vary according to the compartment. For example, H5N1 avian viruses have a tropism for the alpha 2.3 receptors present on the non-ciliated cells of the lower respiratory tract (LRT) and on type II pneumocytes in humans, and are responsible for severe pneumonia (Wang, et al., 2021). In macaques, the H5N1 virus also infects alveolar cell and type II pneumocytes up to day 7 post IT exposure, resulting in high levels of viral replication in the lower respiratory tract and severe disease (Baskin et al., 2009). We show here that radioactive aerosols can help to provide a highly accurate description of the deposition of a biological pathogen, which is likely to affect pathophysiology.

IT is one of the easiest and most used method for RT exposition (Driscoll et al., 2000). Intratracheal instillation bypasses the URT airways eliminating the physical mechanisms naturally involved in particle inhalation. The inoculum is administered in a liquid form, which is influenced by gravity, accounting for the asymmetric and predominantly central lung deposition as observed in this study. Moreover, anatomically, the primary bronchial bifurcation follows a more rectilinear trajectory on the right side than the primary bronchus on the left, potentially accounting for the observed heterogeneous deposition in the lobes of the [¹⁸F]FDG, with more of the liquid deposited within the caudal lobes, and the right caudal lobes in particular. Of note, no such phenomenon was observed after aerosol exposure, although in humans greater deposition would be expected on the left than on the right side (Verbanck et al., 2016). Importantly, we show here that aerosol exposure methods can also influence RT deposition in CM inhalation studies.

Aerosol exposition devices are composed of a generator nebulizing the aerosol, and an interface allowing to present the aerosol to the subject. The 3-jet collision nebulizer is the most widely used nebulizer in aerobiology studies in NHPs. It allows the production of a reproducible and monodisperse aerosol, ideal for targeting the pulmonary alveoli due to its small MMAD (Hartings & Roy, 2004; Zhen et al., 2014). However, jet-nebulization leads to pathogen viability loss (Alsved et al., 2020), requiring very high inoculum doses to compensate for these losses. Mesh nebulizers, such as Aeroneb® Solo device, produces polydisperse aerosols and are an alternative to
<figure><img src="image_17.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_20.png" /></figure>

(caption on next page)
<caption>**Fig. 6.** Radioactive tracking of *Bacillus atrophaeus* in CM after inhalation via a FM.</caption>

a) Chronogram for the macaque inhalation study. CM (n = 3) were exposed by FM inhalation to a 1 mL suspension containing [¹⁸F]FDG (40 MBq) mixed with *Bacillus atrophaeus* spores (10⁸ CFU). The acquisition of positron emission began just after the end of inhalation (10±2min) and continued for 20 min. Sampling was then performed: nasopharyngeal swabs were obtained from both nostrils (NS swabs left and right), together with tracheal swabs and total bronchoalveolar lavage (BAL). Radioactivity was measured with a gamma counter to estimate the number of CFU of *Bacillus atrophaeus* spores in the samples. Samples were stored at +4 °C for 24 h post-sampling (to allow radioactive decay) and the number of CFU actually present was then determined blindly.

b) The calculated CFU is shown on the left for each respiratory tract sample and the real number of CFU is shown on the right. NS swabs left and right are shown in red (circles and triangles, respectively), tracheal swabs are shown as purple diamonds and bronchoalveolar lavages (BAL) are shown as green squares. The black lines represent the geometric means with geometric standard deviations (n = 3 macaques).

c) Weighted correlation between CFU direct quantification of *Bacillus atrophaeus* spores and CFU predicted by radioactivity quantification. Values shown are individual measurement of each sample (BAL, NS swabs left and right, T swabs). The black line corresponds to the correlation line and blue ribbon correspond to the 95 % confidence interval. The equation of the correlation line is indicated with corresponding coefficient of determination R². Weighting was realised with a 1/x² coefficient.

d) The stacked histogram represents the distribution of *Bacillus atrophaeus* spores calculated from PET-CT imaging with [¹⁸F]FDG (proportion of inoculum concentrations) in the respiratory and digestive tracts. Each histogram represents a compartment, with the standard deviation shown in the same color (bars). In red the central lung, in blue the peripheral lung, in light gray the URT and in dark gray the digestive tract. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

maintain viability of fragile agents like viruses during macaque exposure (Bowling et al., 2019). Unlike jet nebulizers, vibrating meshes operate without an external air supply, which limits overpressure inside facemask and associated leakage risk. Moreover, modern mesh vibration frequency controller allows controlled inoculum liquid flow. Therefore, liquid aerosol generation can be configured to limit inoculum losses in the system (Cabrera et al., 2023).

Common aerosol interfaces consist of HOE which stabilise the aerosol concentration in a closed environment. The dynamic aerosol circulation inside the HOE chamber presents well-known challenges in maintaining a spatially uniform particle concentration and especially during spontaneous breathing by NHP (Phalen et al., 1984). A mathematical modeling-based characterization of aerosol concentration dynamics in the HOE is available in the **Supplementary material** to visualize the temporal variation in concentration inside the chamber. However, using an Aeroneb® Solo device, our study shows that HOE results in higher peripheral aerosol deposition than FM exposure in a CM model. This may be explained by the difference in MMAD due to droplets evaporation (1.9 µm versus 3.1 µm, respectively). In contrast, there is neither particle evaporation nor sedimentation with FM (**Supplementary material**). However, our study reveals some limitation of aerosol exposure using HOE. First, the comparison of calculated predicted doses presented (based on Vmin) and the real doses actually deposited revealed considerable variability, which may be problematic in situations in which precise dose accuracy is critical. This can be explained by the anatomical specificities of individuals, such as the minimum cross-section of the nostrils which can partly be taken into account in computational and mathematical modelling of inhalation by NHPS (Kesavan et al., 2020). The position of the animal's head, as well as individual ventilatory parameters and anatomical specificities (flow velocity on inhalation and exhalation, tidal volume and breathing frequency, humidity of the respiratory tract) may lead to differences in deposition during inhalation (Heyder et al., 1986; Martonen et al., 1985). The spray factor measurement using gelatin filters showed reproducibility in aerosol concentration but aerosol is always collected from the same point in the chamber. The chamber's relatively low air renewal (22.5 air changes/hour, meaning a 2.67-min residence time) likely caused variations in particle concentration, especially early in nebulization. Since it took 8 min to reach 95 % equilibrium, these fluctuations, could contribute to the individual deposition variability. Additionally, imaging revealed the deposition of significant amounts of radioactivity on the back of the animals' heads. This phenomenon may be attributed to the HOE's function of aspirating particles to maintain a constant concentration, as the head support grid for the animals is positioned above the aerosol evacuation duct. It is worth noting that 3-dimensional visualization, in contrast to 2-dimensional imaging techniques (e.g., gamma camera), made it possible to separate outside deposition (back of the head, face) from URT deposition. Similarly, we could distinguish the deposition in the esophagus (due to swallowing) compared to the trachea, highlighting the benefit of TEP imaging. The differences in deposition fractions after FM and HOE exposure validates human MMAD-based modelling such as Multi-Path Particle Dosimetry software (MPPD), International Commission on Radiological Protection (ICRP) model or computational fluid dynamics models of inhaled particle deposition (Group of the International Commission on Radiological Protection, 1994; Phalen & Raabe, 2016). Knowing that radioactivity in the digestive tract comes from swallowed particles initially deposited in the upper respiratory tract (URT), we compared our PET/CT results from CM with MPPD calculations (rhesus macaque and human models). We calculated deposition fraction using the two particle sizes: 3.1 µm (GSD 2.2) and 1.9 µm (GSD 2.0) (Supplementary Fig. 4). While our measured deposition fractions for 3.1 µm particles in CM matched MPPD predictions for both rhesus macaques and humans, a different trend was observed for 1.9 µm particles. For these smaller particles, the deposition fraction was similar between the human model and CM, yet diverged from rhesus macaques because of their higher tracheobronchial deposition fraction. This difference might be due to anatomical variations between macaque species, specifically the longer face of CM affecting their respiratory tract shape. These findings underscore the importance of using animal models in inhalation studies.

We propose that the three exposure methods tested here could be used to deliver pathogens according to the specifications of particular scenarios. Pathogen exposure by liquid IT instillation is not an inhalation method and does not mimic natural aerosol exposure. The inoculum is deposited exclusively in the lower respiratory but can be combined with instillation in the nostrils to target the entire RT in the modelling of infectious diseases. It reduces inoculum loss, is easy to control and shows high reproducibility of
exposure regarding total dose, although heterogenous deposition of liquid inoculum within the lungs. The distribution of the inoculum does not follow the physical mechanisms of particle deposition in the respiratory tract during aerosol inhalation. Instillation can be used when animals need to be exposed rapidly after the emergence of a new virus to effectively assess the susceptibility of NHPs. This method is for example valuable for comparing the pathophysiology of H5N1 infection in macaques when the pathogen is introduced solely into the LRT, as opposed to exposure via the nasal route or even just the digestive tract. This distinction is especially relevant given recent publications on the emergence of bovine H5N1 virus clade 2.3.4.4b. (Rosenke et al., 2025). Instillation can therefore be implemented for prophylactic or therapeutic preclinical trials without considering the natural distribution of the pathogen during exposure. This method is of great interest when the pathogen does not target a specific cell type or anatomical structure or when the immune response in upper respiratory tract is not expected to contribute to host defences. On the other hand, aerosol inhalation methods expose both URT and LRT and allows a homogeneous LRT distribution and penetration inside lobes. Aerosol inhalation via a facemask can be used to reproduce exposure to a relatively large, polydisperse aerosol before dehydration, reproducing infectious exposure through relatively close contact like after coughing or sneezing. FM can also be used for aerosol therapy, as it targets the entire tract with high efficacy and limits the inoculum size. Aerosol metrology cannot be performed during inhalation via a facemask, but we show here that [¹⁸F]FDG can be mixed with the pathogen inoculum as a radiotracer to check the deposition of the pathogen dose in the RT, after *in vitro* validation. Alternatively, HOE device can be used to reproduce aerosol exposure inside a closed and poorly ventilated environment. Droplets accumulate in the chamber and are dehydrated by airflow, leading to reduced particle size (<5 µm) that can reach bronchi and alveoli more easily, leading to very homogeneous lung deposits with high particle penetration. This is useful to model pathogens that are highly persistent in the air like influenza or SARS-CoV-2 viruses, mycobacteria or bacterial spores (Fernandez et al., 2019; Smither et al., 2020). Inhaled dose and deposition can be estimated by via aerosol bi collection combined with macaque plethysmography combined with computational modelling like MPPD. However, aerosol exposures are associated to a higher variability in deposited dose and distribution, which need to be taken into account when developing a physio pathological animal model or performing dose effect study. The number of animals used for preclinical studies could be optimized through new approaches, such as imaging analysis, to decrease the degree of uncertainty within experimental groups. This approach corresponds to reduction and refinement, in accordance with the 3R rules.

Two stages in the development of the preclinical infection model could guide the selection of the aerosol exposure method. The first is the determination of the concentration and particle size distribution of the infectious aerosol produced by infected patients, as well as the capacity of the pathogen to survive in aerosol form. This knowledge should guide the method of administration and, in particular, the specificities of the aerosol to which the animal can be exposed that are relevant to natural transmission mechanisms. A scientific review compiles all the physical and biological parameters relevant to the natural transmission of infectious diseases, providing valuable insights to determe appropriate animal exposure methodologies in the laboratory (Pöhlker et al., 2023). The tropism of viruses and, in particular the tissue distribution of entry receptors, can also guide the exposure method, to ensure the targeting of cells responsible for initiating infection, or to influence disease severity by mimicking different exposure scenarios (Kineshta et al., 2025). The second stage consist in the validation of pathophysiology in animals with comparison to that described in human cases. Longitudinal monitoring of pathogen loads kinetic in the different compartments of the RT, combined with clinical signs such as nature and location of lesions as well as development of the immune response should be similar between NHP models and clinical cases, to make it possible to perform therapeutic or prophylactic (e.g. for vaccines) efficacy trials.

Finally, we consider PET/CT to be a useful technology in aerobiology before preclinical trials involving NHP inhalation, for the development of administration techniques for controlling exposure doses. Radiotracking of the pathogen must be validated for each formulation/method of administration and imaging must be performed just after inhalation, to limit the risks of systemic diffusion and clearance of both radiotracers and pathogens.

CRediT authorship contribution statement

**Benoît Delache:** Visualization, Resources, Investigation, Conceptualization, Writing – original draft, Supervision, Methodology, Data curation, Writing – review & editing, Validation, Project administration, Formal analysis. **Anaïs-Rachel Garnier:** Writing – review & editing, Validation, Investigation, Conceptualization, Visualization, Methodology, Data curation, Writing – original draft, Software, Formal analysis. **Cécile Herate:** Writing – original draft, Project administration, Formal analysis, Writing – review & editing, Validation, Investigation, Conceptualization, Visualization, Methodology, Data curation. **Francis Relouzat:** Writing – review & editing, Supervision, Methodology, Validation, Project administration, Visualization, Resources, Conceptualization. **Pierre Le-Bury:** Writing – review & editing, Validation, Formal analysis, Visualization, Methodology, Writing – original draft, Software, Data curation. **Julien Lemaitre:** Visualization, Formal analysis, Writing – original draft, Methodology, Conceptualization, Writing – review & editing, Software, Data curation. **Asma Berriche:** Methodology, Conceptualization, Software, Formal analysis, Writing – review & editing, Investigation. **Quentin Sconosciuti:** Writing – review & editing, Conceptualization, Investigation, Methodology. **Eleana Navarre:** Methodology, Writing – review & editing, Formal analysis, Investigation. **Noémie Verguet:** Investigation, Methodology, Writing – review & editing, Data curation. **Justina Creppy:** Investigation. **Olivier Gorgé:** Resources, Funding acquisition, Writing – review & editing, Investigation, Conceptualization, Methodology, Data curation. **Jean-Nicolas Tournier:** Writing – review & editing, Investigation, Resources, Supervision, Funding acquisition. **Frédéric Ducancel:** Resources, Investigation, Supervision, Methodology, Conceptualization, Writing – review & editing, Project administration, Funding acquisition. **Laurent Vecellio:** Validation, Resources, Writing – original draft, Software, Investigation, Conceptualization, Writing – review & editing, Supervision, Methodology, Data curation, Formal analysis. **Roger Le Grand:** Supervision, Methodology, Conceptualization, Validation, Project administration, Funding acquisition, Writing – review & editing, Resources, Investigation. **Thibaut Naninck:** Validation, Resources, Funding
acquisition, Conceptualization, Visualization, Software, Investigation, Data curation, Writing – review & editing, Supervision, Methodology, Formal analysis.

Declaration of competing interest

The authors declare no competing interests.

Acknowledgements

We would particularly like to thank the Infectious Disease Models and Innovative Therapies research infrastructure (IDMIT) for its
important support for the *in vitro* and *in vivo* experiments performed in this study. We thank all members of the Animal Science and
Welfare and Imaging of Infection and Immunity core facilities for their excellent expertise and outstanding contributions: Sebastien
Langlois, Maxime Potier, Jean-Marie Robert, Victor Magneron, Emma Burban, Flavie Misplon, Eleonore Sizun, Tatiana Geara. We
thank the Uniivo team for their excellent care of the animals. We thank Sylvie Legendre, Yann Gorin, Alicia Pouget and Sebastien
Jacquin for support and expertise in the transport and manipulation of biological agents and radiological security. We thank Maria
Cabrera for her expertise and advice on scintigraphy and nuclear imaging. We thank the LIFEx software developers. IDMIT is supported
by the "Programme Investissements d'Avenir" (PIA), managed by the ANR under references ANR-11-INBS-0008 and ANR-10-EQPX-
02-01. This research was funded by the French interministerial program for research and development in the fight against CBRN
(Chemical, Biological, Radiological, and Nuclear) threats and the RESILIENCE-R-2023 project (Grant agreement n. 101168024) of the
European Defence Fund. Views and opinions expressed are those of the author(s) and do not necessarily reflect those of the European
Union or the European Defence Fund. Neither the European Union nor the granting authority can be held responsible for them. Justina
Creppy PhD grant has been funded by CEA- Infectious Disease Models and Innovative Therapies (IDMIT), Fontenay-aux Roses, France
and the Agence Innovation Défense (AID) France.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jaerosci.2025.106646.

Data availability

Data will be made available on request.

References

2024 global tuberculosis report. (2024).

Alsved, M., Bourouiba, L., Duchaine, C., Löndahl, J., Marr, L. C., Parker, S. T., ... Thomas, R. J. (2020). Natural sources and experimental generation of bioaerosols: Challenges and perspectives. *Aerosol Science and Technology*, *54*, 547–571.

Bartrand, T. A., Weir, M. H., & Haas, C. N. (2008). Dose-response models for inhalation of *Bacillus anthracis* spores: Interspecies comparisons. *Risk Analysis*, *28*, 1115–1124.

Baskin, C. R., Bielefeldt-Ohmann, H., Tumpey, T. M., Sabourin, P. J., Long, J. P., García-Sastre, A., ... Katze, M. G. (2009). Early and sustained innate immune response defines pathology and death in nonhuman Primates infected by highly pathogenic influenza virus. *Proceedings of the National Academy of Sciences of the United States of America*, *106*, 3455–3460.

Beck, S. E., Laube, B. L., Barberena, C. I., Fischer, A. C., Adams, R. J., ... Chesnut, K. (2002). Deposition and expression of aerosolized rAAV vectors in the lungs of rhesus macaques. *Molecular Therapy*, *6*, 546–554.

Bowling, J. D., O'Malley, K. J., Klimstra, W. B., Hartman, A. L., & Reed, D. S. (2019). A vibrating mesh nebulizer as an alternative to the collision three-jet nebulizer for infectious disease aerobiology. *Applied and Environmental Microbiology*, *85*.

Bixler, S. L., Stefan, C. P., Jay, A. N., Rossi, F. D., Ricks, K. M., ... Shoemaker, C. J. (2022). Exposure route Influences Disease Severity in the COVID-19 Cynomolgus Macaque Model. *Viruses*, *14*, 1013.

Boydston, J. A., Biryukov, J., Yeager, J. J., Zimmerman, H. A., Williams, G., Green, B., ... Dabisch, P. A. (2023). Aerosol particle size influences the infectious dose and disease severity in a golden Syrian hamster model of inhalational COVID-19. *Journal of Aerosol Medicine and Pulmonary Drug Delivery*, *36*, 235–245.

Brand, P., Schulte, M., Wencker, M., Herpich, C. H., Klein, G., Hanna, K., & Meyer, T. (2009). Lung deposition of inhaled α1-proteinase inhibitor in cystic fibrosis and α1-antitrypsin deficiency. *European Respiratory Journal*, *34*, 354–360.

Cabrera, M., Le Pennec, D., Le Guellec, S., Pardessus, J., Ehrmann, S., MacLoughlin, R., ... Vecellio, L. (2023). Influence of mesh nebulizer characteristics on aerosol delivery in non-human Primates. *European Journal of Pharmaceutical Sciences*, *191*, Article 106606.

Creppy, J. R., Delache, B., Lemaitre, J., Horvat, B., Vecellio, L., & Ducancel, F. (2024). Administration of airborne pathogens in non-human primates. *Inhalation Toxicology*, *36*, 475–500.

Dabisch, P. A., Xu, Z., Boydston, J. A., Solomon, J., Bohannon, J. K., Yeager, J. J., ... Lackemeyer, M. G. (2017). Quantification of regional aerosol deposition patterns as a function of aerodynamic particle size in rhesus macaques using PET/CT imaging. *Inhalation Toxicology*, *29*, 506–515.

Day, W. C., & Berendt, R. F. (1972). Experimental tularemia in *Macaca mulatta*: relationship of aerosol particle size to the infectivity of airborne Pasteurella tularensis. *Infection and Immunity*, *5*, 77–82.

Dolovich, M. B. (2001). Measuring total and regional lung deposition using inhaled radiotracers. *Journal of Aerosol Medicine*, *14*, S35–S44.

Dolovich, M. B., & Bailey, D. L. (2012). Positron emission tomography (PET) for assessing aerosol deposition of orally inhaled drug products. *Journal of Aerosol Medicine and Pulmonary Drug Delivery*, *25*, S52-71.

Druett, H. A., Robinson, J. M., Henderson, D. W., Packman, L., & Peacock, S. (1956). Studies on respiratory infection: II. The influence of aerosol particle size on infection of the guinea-pig with Pasteurella pestis. *Journal of Hygiene*, *54*, 37–48.

Fennelly, K. P. (2020). Particle sizes of infectious aerosols: Implications for infection control. *Lancet Respiratory Medicine*, *8*, 914–924.
Fernandez, M. O., Thomas, R. J., Garton, N. J., Hudson, A., Haddrell, A., & Reid, J. P. (2019). Assessing the airborne survival of bacteria in populations of aerosol droplets with a novel technology. *Journal of The Royal Society Interface, 16*, Article 20180779.

Driscoll, K. E., Costa, D. L., Hatch, G., Henderson, R., Oberdorster, G., Salem, H., & Schlesinger, R. B. (2000). Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: Uses and limitations. *Toxicological Sciences, 55*, 24–35.

Fleming, J. S., Halson, P., Conway, J., Moore, E., Nassim, M. A., Hashish, A. H., ... Martonen, T. B. (1996). Three-dimensional description of pulmonary deposition of inhaled aerosol using data from multimodality imaging. *Journal of Nuclear Medicine, 37*, 873–877.

Gardner, M. B., & Luciw, P. A. (2008). Macaque models of human infectious disease. *ILAR Journal, 49*, 220–255.

Group of the International Commission on Radiological Protection. (1994). Human respiratory tract model for radiological protection. *Annals of the ICRP, 24*(1–3), 1–482.

Hartings, J. M., & Roy, C. J. (2004). The automated bioaerosol exposure system: Preclinical platform development and a respiratory dosimetry application with nonhuman primates. *Journal of Pharmacological and Toxicological Methods, 49*, 39–55.

Heyder, J., Gebhart, J., Rudolf, G., Schiller, C. F., & Stahlhofen, W. (1986). Deposition of particles in the human respiratory tract in the size range 0.005-15 µm. *Journal of Aerosol Science, 17*, 811–825.

Kanekiyo, M., Gillespie, R. A., Midgett, M., O'Malley, K. J., Williams, C., Moin, S. M., ... Reed, D. S. (2023). Refined semi-lethal aerosol H5N1 influenza model in cynomolgus macaques for evaluation of medical countermeasures. *iScience, 26*, Article 107830.

Hofer, S., Hofstätter, N., Duschl, A., & Himly, M. (2021). SARS-CoV-2-laden respiratory aerosol deposition in the lung alveolar-interstitial region is a potential risk factor for severe disease: A modeling study. *Journal of Personalized Medicine, 11*, 431.

Kesavan, J. S., Alstadt, V. J., & Laube, B. L. (2020). Aerosol deposition in 3D models of the upper airways and trachea of rhesus macaques. *Aerosol Science and Technology, 54*, 983–991.

Kineshta, P., Marr, L. C., Henriques, A., Martin, A. R., Prussin, A. J., Aleixo, L., ... Tang, J. W. (2025). Can we use viral receptor mapping and particle deposition models to predict the clinical severity of novel airborne pathogens? *Clinical Microbiology and Infections, 50*, 503–505.

Lee, Z., & Berridge, M. S. (2002). PET imaging-based evaluation of aerosol drugs and their delivery devices: Nasal and pulmonary studies. *IEEE Transactions on Medical Imaging, 21*, 1324–1331.

Lemaitre, J., Naninck, T., Delache, B., Creppy, J., Huber, P., Holzapfel, M., ... Maisonnasse, P. (2021). Non-human primate models of human respiratory infections. *Molecular Immunology, 135*, 147–164.

Leung, N. H. L. (2021). Transmissibility and transmission of respiratory viruses. *Nature Reviews Microbiology, 19*, 528–545.

Li, Y. (2021). Basic routes of transmission of respiratory pathogens—A new proposal for transmission categorization based on respiratory spray, inhalation, and touch. *Indoor Air, 31*, 3–6.

Li, H., Leong, F. Y., Xu, G., Kang, C. W., Lim, K. H., Tan, B. H., & Loo, C. M. (2021). Airborne dispersion of droplets during coughing: a physical model of viral transmission. *Scientific Reports, 11*, 4617.

Madas, B. G., Füri, P., Farkas, A., Nagy, A., Czitrovszky, A., Balásházy, I., ... Horváth, A. (2020). Deposition distribution of the new coronavirus (SARS-CoV-2) in the human airways upon exposure to cough-generated aerosol. *Scientific Reports, 10*, 22430.

Mairinger, S., Hernández-Lozano, I., Zeitlinger, M., Ehrhardt, C., & Langer, O. (2022). Nuclear medicine imaging methods as novel tools in the assessment of pulmonary drug disposition. *Expert Opinion on Drug Delivery, 19*, 1561–1575.

Marriott, A. C., Dennis, M., Kane, J. A., Gooch, K. E., Hatch, G., Sharpe, S., ... Carroll, M. W. (2016). Influenza A virus challenge models in cynomolgus macaques using the authentic inhaled aerosol and intra-nasal routes of infection. *PLOS One, 11*, Article e0157887.

Martonen, T. B., Barnett, A. E., & Miller, F. J. (1985). Ambient sulfate aerosol deposition in man: Modeling the influence of hygroscopicity. *Environmental Health Perspectives, 63*, 11–24.

Memoli, M. J., Shaw, P. A., Han, A., Czajkowski, L., Reed, S., Athota, R., ... Taubenberger, J. K. (2016). Evaluation of antihemagglutinin and antineuraminidase antibodies as correlates of protection in an influenza A/H1N1 virus healthy human challenge model. *mBio, 7*, Article e00417, 16.

Miller, L. A., Royer, C. M., Pinkerton, K. E., & Schelegle, E. S. (2017). Nonhuman primate models of respiratory disease: Past, present, and future. *ILAR Journal, 58*, 269–280.

Mooij, P., et al. (2021a). Oral, and nasal inoculation in cynomolgus macaques. *Viruses, 13*, 345.

Mooij, P., et al. (2021b). Aerosolized pH1N1 influenza infection induces less systemic and local immune activation in the lung than combined intrabronchial, nasal and oral exposure in cynomolgus macaques. *Journal of General Virology, 101*, 1229–1241.

Nioche, C., Orlhac, F., Boughdad, S., Reuzé, S., Goya-Outi, J., Robert, C., ... Buvat, I. (2018). Lifex: A freeware for radiomic feature calculation in multimodality imaging to accelerate advances in the characterization of tumor heterogeneity. *Cancer Research, 78*, 4786–4789.

Phalen, R. F., Mannix, R. C., & Drew, R. T. (1984). Inhalation exposure methodology. *Environmental Health Perspectives, 56*, 23–34.

Phalen, R. F., Oldham, M. J., & Wolff, R. K. (2008). The relevance of animal models for aerosol studies. *Journal of Aerosol Medicine and Pulmonary Drug Delivery, 21*, 113–124.

Phalen, R. F., & Raabe, O. G. (2016). The evolution of inhaled particle dose modeling: A review. *Journal of Aerosol Science, 99*, 7–13.

Pöhlker, M. L., Pöhlker, C., Krüger, O. O., Förster, J. D., Berkemeier, T., Elbert, W., ... Mikhailov, E. (2023). Respiratory aerosols and droplets in the transmission of infectious diseases. *Reviews of Modern Physics, 95*.

Rosenke, K., Giffin, A., Kaiser, F., Altynova, E., Mukesh, R., ... Bushmaker, T. (2025). Pathogenesis of bovine H5N1 clade 2.3.4.4b infection in macaques. *Nature, 640*, 1017–1021.

Roy, C. J., Reed, D. S., & Hutt, J. A. (2010). Aerobiology and inhalation exposure to biological select agents and toxins. *Veterinary Pathology Online, 47*, 779–789.

Smither, S. J., Eastaugh, L. S., Findlay, J. S., & Lever, M. S. (2020). Experimental aerosol survival of SARS-CoV-2 in artificial saliva and tissue culture media at medium and high humidity. *Emerging Microbes and Infections, 9*, 1415–1417.

Verbanck, S., Ghorbaniasl, G., Biddiscombe, M. F., Dragojlovic, D., Ricks, N., Lacor, C., ... Usmani, O. S. (2016). Inhaled aerosol distribution in human airways: A scintigraphy-guided study in a 3D printed model. *Journal of Aerosol Medicine and Pulmonary Drug Delivery, 29*, 525–533.

Wang, C. C., et al. (2021a). Airborne transmission of respiratory viruses. *Science (80), 373*.

Wang, D., Zhu, W., Yang, L., & Shu, Y. (2021). The epidemiology, virology, and pathogenicity of human infections with avian influenza viruses. *Cold Spring Harbor Perspectives in Medicine, 11*, Article a038620.

Wonderlich, E. R., Swan, Z. D., Bissel, S. J., Hartman, A. L., Carney, J. P., O'Malley, K. J., ... Barratt-Boyes, S. M. (2017). Widespread virus replication in alveoli drives acute respiratory distress syndrome in aerosolized H5N1 influenza infection of macaques. *The Journal of Immunology, 198*, 1616–1626.

Yan, J., Grantham, M., Pantelic, J., Bueno De Mesquita, P. J., Albert, B., Liu, F., ... Emit, Consortium. (2018). Infectious virus in exhaled breath of symptomatic seasonal influenza cases from a college community. *Proceedings of the National Academy of Sciences of the United States of America, 115*, 1081–1086.

Zhen, H., Han, T., Fennell, D. E., & Mainelis, G. (2014). A systematic comparison of four bioaerosol generators: Affect on culturability and cell membrane integrity when aerosolizing Escherichia coli bacteria. *Journal of Aerosol Science, 70*, 67–79.

Zhou, M., & Zou, J. (2021). A dynamical overview of droplets in the transmission of respiratory infectious diseases. *Physics of Fluids, 33*, 1–38.