**1. Introduction**

Several infectious diseases are known to be transmissible through the airborne route, such as tuberculosis and measles. The only known disease transmitted only through the airborne route is tuberculosis, as reported by Roy and Milton [1]. Aerosol transmission of other diseases could be preferential or opportunistic [1]. Therefore, it is difficult to assess the importance (or not) of the airborne route in disease transmission. The mode of transmission of some diseases is ambiguous. The evidence of severe acute respiratory syndrome (SARS) airborne transmission was first assessed by indirect evidences such as modeling and epidemiological studies [2]. Moreover, the possible airborne transmission of non-respiratory diseases, like Norovirus, is a subject of investigation [3–5].

For many diseases, dissociating transmission routes such as indirect contact, exposure to large droplets and aerosol transmission through aerosols can be complicated, even in controlled laboratory environments. The World Health Organization considers disease transmission with particles >5 μm as droplets transmission and with particles <5 μm as aerosols transmission [6].

The size of the particles involved in the natural transmission of diseases through the airborne route is hard to establish, especially for viral diseases. In fact, only a few studies have looked at the particle size of airborne viruses that can be found in the environment. Anderson 6 stage cascade impactors, National Institute for Occupational Safety and Health (NIOSH) two-stage bioaerosols cyclone samplers [7–9], and Sioutas personal cascade impactors [10,11] have been used in agricultural and hospital settings. In all these studies, viruses were found in all air sample stages, meaning that large particles as well as small particles can carry viruses. More recently, in a laboratory setting, experiments using ferrets and particle impactors of various cut-off sizes demonstrated that influenza virus can be transmitted via droplets (15.3–5 μm) as well as airborne particles (5–1.5 μm) [12].

Information on the infectious state of airborne viruses is sparse [13]. Culture on appropriate cell lines is still the gold standard to assess virus infectivity. However, the culture of airborne viruses faces several challenges: (1) low concentrations of viruses in the air require large air volume sampling to allow detection (meaning extensive air sampling periods or the use of high-flow air samplers); (2) viruses can be damaged during air sampling; (3) environmental contaminants can interfere with virus or host cell growth (bacteria, mold, dust, etc.).

The use of animals in laboratory settings can overcome most of these challenges. The virus source can be a sick human, an infected animal, or an artificially generated aerosol. By exposing animals to airborne viruses, air sampling can be avoided (preventing virus damage) as well as laboratory virus culture bias in detection. As an example, using animals instead of air samplers can lead to the demonstration that airborne viruses can [14] or cannot [15] infect healthy animals and also that airborne viruses can remain (or not) infectious long enough to travel to a new host. Using a sick animal or human as an aerosol source has also demonstrated that a sick subject can emit aerosols that can potentially infect other susceptible hosts [12].

Unfortunately, the exposure of healthy animals to aerosols emitted by another animal over several days cannot be performed in commercially available apparatus settings. Indeed, cages designed for animal aerosol exposure are meant for a few minutes per day exposure and cannot be used for housing animals for several days. In contrast, animal cages designed to house subjects over extended periods of time are not airtight, and therefore provide limited information about airborne transmission. These cages can be used to prevent direct contact between index and healthy animals and can be placed at various distances but cannot control the size of particles traveling between cages.

In this study, we designed, constructed and tested a system composed of three airtight cages to study the transmission of infectious agents between animals through large droplets and through airborne particles. The system can house three ferrets for up to 10–12 days and is designed to prevent direct contact between animals. We designed a particle separator to prevent large droplets transmission between cages. The cage system is under negative pressure, with high-efficiency particulate air (HEPA) filters on the air inlet and outlet for the users' and environment's protection. This communication describes the main components of this cage system, the particle separator validation using standard aerosol generators as well as a test trial with ferrets and the influenza virus.
