Bioaerosols in swine confinement buildings : nature, fate, and mitigation

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© Jonathan Vyskocil, 2021

Bioaerosols in Swine Confinement Buildings : Nature,

Fate, and Mitigation

Thèse

Jonathan Vyskocil

Doctorat en microbiologie

Philosophiæ doctor (Ph. D.)

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Bioaerosols in Swine Confinement Buildings:

Nature, Fate, and Mitigation

Thèse

Jonathan Mark Vyskocil

Sous la direction de :

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Résumé

L’industrialisation du secteur porcin canadien a menée à une diminution du nombre de fermes et une augmentation de porc par bâtiment avec comme conséquence, un risque accru d’épidémies. Les mesures de biosécurité appliquées n’incluent pas d’exigences pour le contrôle des bioaérosols (voies de transmission confirmée ou suspectée pour plusieurs maladies). Certains pathogènes bactériens et viraux peuvent parcourir de longues distances dans l’air et garder leur potentiel infectieux. La recherche de méthodes visant à contrôler les bioaérosols est active et l’utilisation de technologies fiables et abordables de traitement de l’air amélioreraient le bien-être animal et pourraient réduire l’incidence des épidémies et les coûts conséquents pour l’industrie.

Lors de ce programme doctoral, quatre types de traitement de l’air ont été examinés, un biofiltre percolateur, l’ozone dans un tunnel, des filtres mécaniques et des filtres antimicrobiens. Différents modèles d’exposition aux bioaérosols ont été testés comme une aérosolisation sèche ou liquide, d’origine naturelle ou artificielle. En outre, une méthode de production de poussière contaminée par des virus infectieux a été développée.

Dans la première étude, un tunnel de vent a été utilisé pour aérosoliser et exposer le phage PhiX174 à l’ozone à des concentrations variables et deux conditions humidités relatives (40% et 80%). L’ozone a été généré à 0,3 à 1,8 ppm en comparaison avec des expériences sans ozone pour évaluer la perte des phages aérosolisés dans le tunnel. Les phages ont été collectés en solution liquide par deux collecteurs simultanément avec un point de référence en amont de l’ajout d’ozone (ou temps de référence zéro). Les phages ont été quantifiés par qPCR et par culture pour établir les ratios infectieux. À chaque point de prélèvement, le ratio total/infectieux a été comparé à celui au temps zéro. Les résultats de l’étude ont été difficiles à interpréter à cause d’un effet de l’ozone sur les phages dans les récipients de collecte. Ce phénomène a mené à l’application d’un facteur de correction dans les résultats observés. Après correction, une diminution significative entre les ratios a été observée entre 0, 0,3 et 0,6 ppm à 80% d’humidité relative tandis qu’aucune différence n’a été observée entre les concentrations à 40% d’humidité.

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Dans un deuxième temps, des bioaérosols artificiels ont été produits par aérosolisation sèche pour étudier leur utilité dans l’évaluation de l’efficacité de réduction des filtres mécaniques et antimicrobiens. Aucune étude à ce jour ne proposait de protocole afin de produire des aérosols viraux secs de distribution granulométrique similaire à celle rencontrée dans le milieu porcin. Un tel aérosol permettrait l’obtention de conditions expérimentales plus représentatives de l’environnement étudié car les aérosols liquides créés en laboratoire sont habituellement plus petits. Nous avons produit des bioaérosols secs de phages infectieux agrégés sur une poussière standard de diamètre aérodynamique médian en masse de 12 μm. Le protocole a permis la préparation de poussière standard contenant des phages (PhiX174 ou MS2) mais seul MS2 a montré une concentration assez élevée pour être détectée après filtration mécanique et antimicrobienne (PhiX174 a été réduit en-dessous de la limite de détection). Cette méthode pourra être appliquée à d’autres types de filtres.

Finalement, un biofiltre percolateur traitant l’air d’une salle d’engraissement d’une porcherie a été évalué pour la réduction des bactéries, archées, et virus émis par les systèmes de ventilation du bâtiment. Ce projet a évalué l’impact du taux de ventilation et des températures extérieures sur l’efficacité du système percolateur grâce à une série d’échantillonnages échelonnés sur une période de 10 mois. L’effet potentiel du biofiltre percolateur comme source d’émission de bioaérosols a été étudié par analyse de biodiversité par séquençage à haut débit tandis que l’efficacité de la réduction microbienne des filtres a été testée par qPCR. Cette étude a montré que le contenu de l’air émis par le biofiltre percolateur était différent en composition de celle des échantillons d’air en amont. Les résultats de qPCR ont montré des efficacités de réduction des bioaérosols modérées à élevées pour les bactéries et les archées. Les coliphages, seuls virus aéroportés détectés dans l’air sortant de la porcherie, n’ont été réduits que faiblement, tout comme les bactéries cultivables. Aucun effet de la température extérieure n’a été identifié au niveau de l’efficacité de la réduction microbienne.

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Abstract

In Canada, the number of pigs per farm has increased while the number of farms decreases, largely due to the shift to industrialized agriculture. The large number of animals in buildings increases the potential costs due to outbreaks as more animals can be exposed to pathogens. Therefore, biosecurity measures are extremely important to prevent disease introduction and spread. Many modern biosecurity standards have no requirements over the control of bioaerosols, a potential transmission pathway. Some bacterial and viral pathogens have been shown to be able to travel long distances through the air and maintain their infectious potential. Research of methods for reducing bioaerosols is ongoing and many methods proposed for the treatment of undesirable gases have been examined for their bioaerosols reduction. The use of an air treatment technology able to remove multiple airborne contaminants (gases, odors, bioaerosols) reliably and affordably would greatly improve the animal well-being and provide a cost-saving technology for industry operators.

During this doctoral program, four types of air treatment technologies were examined, a percolating biofilter, an ozone treatment system, mechanical filters, and antimicrobial filters. Different modes of bioaerosols exposure were used, naturally occurring bioaerosols from an active swine building and artificially generated, dry and wet, aerosolizations.

In the first study, a wind tunnel system was used to expose nebulized PhiX174 bacteriophages (or phages) to ozone at varying concentrations under two relative humidity (40% and 80%). Ozone was generated at 0.3 to 1.8 ppm at 0.3 ppm increments and included experiments with no ozone to evaluate and adjust results according to possible losses of aerosolized phages in the tunnel. Phages were collected in liquid solutions by two AGI-30 operating simultaneously at two of the five available locations, including a sampling port located before the introduction of ozone. Phages were quantified by qPCR and cultures to establish infectious ratios. The infectious ratio at each time point was compared to the infectious ratio at time point 0. The results of the study were initially confounded by the early discovery of an apparent effect of ozone on phages in the collection recipients. This was accounted for through experiments exposing phage-spiked liquid solutions to the wind tunnel and ozone to measure the effect of ozone on phages in recipients. After corrections, no

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significant difference between any concentrations and 0 ppm ozone could be found at 40% relative humidity. At 80% relative humidity, significant decreases in ratios were observed between 0, 0.3, and 0.6 ppm, while no significant effect of time was observed at either relative humidity.

Secondly, artificial dry bioaerosols were produced and aerosolized to prove their usefulness in evaluating filter reduction efficiencies. No known study has provided a method of generating viral dry bioaerosols of phages aggregated onto dusts of known size (standard) that could provide an artificial bioaerosol more reflective of specific environments. Here, we produced dry bioaerosols of phages aggregated onto a standard test dust with an approximate mass median aerodynamic diameter (MMAD) of 12μm. While both PhiX174 and MS2 phages are able to produce phage-dust aggregates, only MS2 phages had high enough concentrations to be detected following filtration by mechanical and antimicrobial filters while PhiX174 was reduced below detection limit by mechanical filters. This study adds the knowledge of the reduction efficiency of the antimicrobial and mechanical filters against aerosolized artificial bioaerosols dust with an MMAD similar to swine building aerosols as well as proposing a method to produce phage-bearing dusts to challenge such filters.

Lastly, a percolating biofiler treating air from the finishing room of an operational swine confinement building was evaluated for the reduction of bacteria, archaea, and viruses through qPCR and culture analyses. This project evaluated potential changes in reduction related to ventilation rates and temperature by sampling over a 10 month period. Previous concerns of percolating biofilters acting as sources of bioaerosols was examined through diversity analyses. In this study, the nutritive solution bacterial community was different from air samples. Overall, qPCR results showed moderate to good reduction efficiencies for bacteria and archaea, while coliphages, the only detectable viruses in air samples, had a low reduction efficiency. Culturable bacteria also exhibited low reduction efficiencies although there was large variation in the data. No relationship between temperature and bioaerosols concentration could be identified.

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List of Figures

Figure 0-1 Examples of A) positive and B) negative-pressure ventilation systems. ... 3

Figure 0-2 Diagram of air scrubber systems including acid and biological scrubbers. ... 13

Figure 0-3 The ASHRAE 52.2 standard test duct diagram. ... 22

Figure 1-1 The wind tunnel system. ... 35

Figure 1-2 The infectious ratio at time points as a ratio to no exposure. ... 40

Figure 2-1 Diagram of the test duct used in experiments, based from the ASHRAE Standard 52.2. ... 53

Figure 2-2 Preservation of infectivity of PhiX174 and MS2 phages with test dust and cryoprotectants after lyophilization. ... 59

Figure 2-3 TEM imaging of lyophilized mixture of MS2 phages – ISO 12103-1 A3 medium test dust. ... 60

Figure 2-4 The total number of dust particles by ELPI stage. ... 62

Figure 2-5 Total MS2 phages (copies) per cm3_{of air per stage of ELPI. ... 63}

Figure 3-1 Percent reduction efficiencies of ATU against all detectable studied microorganisms. ... 87

Figure 3-2 Dendrogram of samples. ... 89

Figure 3-3 Relative abundances of phyla in samples. ... 90

Figure 4-1 Bacterial diversity analyses of the samples from the first two visits for the percolating biofilter project... 103

Figure 4-2 PCoA plot of concentrated samples... 104

Figure 4-3 Swine buildings and the installation of air treatment technology. ... 107

Figure A-1 Effect of ozone concentrations on phage genomes compared to culturable phages. ... 130

Figure B-1 The SAG 410 connected to the ASHRAE 52.2 Standard-inspired wind tunnel. ... 131

Figure B-2 Samplers used connected to the wind tunnel. ... 131

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List of Tables

Table 0-1 Minimum Efficiency Reporting Value Parameters. ... 22 Table 2-1 Airflow and environmental conditions inside the test duct during ELPI measuring and sampling of artificial viral aerosols. ... 61 Table 2-2 Airflow and environmental conditions inside the test duct during the challenge of MERV-16 and antimicrobial filters with artificial viral aerosols. ... 64 Table 2-3 Reduction efficiencies of MERV-16 and antimicrobial filters. ... 65 Table 3-1 qPCR protocols for targeted microorganisms. ... 81 Table 3-2 Table of recorded air treatment unit (ATU) environmental conditions during sampling. ... 84 Table 3-3 Percent reduction efficiencies for analyses by grouping. ... 88

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List of Abbreviations

AGI ... All-Glass Impinger

APP ... Actinobacillus pleuropneumoniae

ASHRAE ... American Society of Heating, Refrigeration, and Air-Conditioning Engineers

BEV ... Bovine enterovirus 1 CFU ... Colony forming units

CSHB ... Canadian Swine Health Board DNA ... Deoxyribonucleic acid

EAV ... Equine arteritis virus

ELPI ... Electrical Low-Pressure Impactor FISH ... Fluorescent in situ hybridization HEPA ... High-Efficiency Particulate Air

HTAS ... High-throughput amplicon sequencing IAV ... Influenza A virus

MALDI-TOF ... Matrix-associated laser desorption/ionization and mass analyzer is time-of-flight

MERV ... Minimum efficiency reporting value MMAD ... Mass median aerodynamic diameter MPN ... Most probable number

MRSA ... Methicillin resistant Staphylococcus aureus MS ... Mass spectrometry

OPS ... Optical particle sizer PCR ... Polymerase chain reaction PCV ... Porcine circovirus

PEDV ... Porcine epidemic diarrhea virus PM ... Particulate matter

ppm ... Parts per million

PRRSV ... Porcine reproductive and respiratory syndrome virus qPCR ... Quantitative polymerase chain reaction or real-time PCR RNA ... Ribonucleic acid

rRNA ... Ribosomal ribonucleic acid SAG ... Solid Aerosol Generator SSPD ... Small-scale powder disperser TSA ... Trypticase soy agar

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Acknowledgements

I would like to begin by thanking my supervisor, Dr. Caroline Duchaine, for the opportunity to explore and study the field of bioaerosols. This was a new field of research for me and she supported me as I learned many new aspects of microbiology relating to bioaerosols. I am grateful for her encouragement for both advancing my knowledge, skills, and practicing French.

I am also extremely grateful to Valerie who was charged with supervision of the majority of my projects. I enjoyed our discussions during the travels to sites. She instructed me on the methods used in the lab and the methods of sampling bioaerosols. She was a fantastic supervisor and colleague who helped me in all aspects of my program. If I was overwhelmed with work she was willing to assist me with work to ensure it is completed in a timely manner. I would like to mention Jodelle for her consistent greetings every day, encouraging me to practice conversational French, board game nights, and conversations at work. Of course, the entirety of the Duchaine Bioaerosols Group is to thank for their collaboration and conversations as well.

I need to thank the collaborators from IRDA and CDPQ for their work on the projects, setting up and providing access to the testing systems, wind tunnels, and air treatment technologies. This work could not have been done without them.

I would like to thank the funding agencies who helped fund this doctoral work, the Centre de recherche en infectiologie porcine et avicole (CRIPA) and the Quebec Respiratory Health Network for their provisions of scholarships and travel grants so I can learn new skills in different research groups. As well, I would like to thank for the Université Laval for their scholarships that they provide graduate students during various points in their academic careers.

I would like to finally thank the members of my jury for agreeing to be on my committee and reviewing my doctoral work, presented in this thesis.

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Foreword

This thesis discusses the outcome of the three projects of this doctoral program exploring treatment strategies to reduce bioaerosols of swine buildings and alternative means of testing reduction strategies with a representative standardized artificial bioaerosol mixture. This document opens with an introduction to the topics involved with this work, a brief summary of the swine industry and its importance in Canada, biosecurity considerations for the industry, and bioaerosols – specifically bioaerosols that originate from swine and are of particular concern to the industry – and their transmission, methodology of evaluation, and applied or experimental reduction strategies. Following the introduction are the inserted articles from this work, Chapter 1 – Ozone treatment in a wind tunnel for the reduction of airborne viruses in swine buildings, Chapter 2 – Challenge of mechanical and antimicrobial filters against infectious phages artificially agglomerated with inorganic dust with a known particle-size distribution, and Chapter 3 – Reduction of bioaerosols emitted from a swine confinement building by a percolating biofilter during a 10-month period. At the end of this document is the overall discussion and conclusion of this work summarizing all the projects and describing how this body of work connects to existing knowledge.

Chapter 1 – Ozone treatment in a wind tunnel for the reduction of airborne viruses in swine buildings, as of writing, has been published in Aerosol Science and Technology. Vyskocil, J. M., Turgeon, N., Turgeon, J. G., & Duchaine, C. (2020). Ozone treatment in a wind tunnel for the reduction of airborne viruses in swine buildings. Aerosol Science and

Technology, 54(12), 1471-1478.

Chapter 2 – Challenge of mechanical and antimicrobial filters against infectious phages artificially agglomerated with inorganic dust with a known particle-size distribution was published in Aerosol Science and Technology. Vyskocil, J. M., Létourneau, V., St– Germain, M. W., Turgeon, J. G., & Duchaine, C. (2020). Challenge of mechanical and antimicrobial filters against infectious phages artificially agglomerated with inorganic dust with a known particle-size distribution. Aerosol Science and Technology, 1-11.

Chapter 3 –Reduction of bioaerosols emitted from a swine confinement building by a percolating biofilter during a 10-month period was published in Atmosphere and this is the

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version included. The citation for this article is: Vyskocil, J. M., Létourneau, V., Girard, M., Lévesque, A., & Duchaine, C. (2019). Reduction of Bioaerosols Emitted from a Swine Confinement Building by a Percolating Biofilter During a 10-Month Period. Atmosphere, 10(9), 525.

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Introduction

Swine Industry in Canada

The Canadian swine industry contributes significantly to global pork exports, approximately 1.4 million metric tons as of January 2020 (USDA-FAS 2020). In January 2019, Quebec housed the greatest number of hogs of all provinces in Canada, 4.385 million heads of the 13.975 million heads in Canada (Statistics Canada 2020a). The stability of the swine industry is a concern nationally as well as provincially. The swine industry has transitioned into a heavily industrialized sector and specialized operations as fewer farms with greater number of pigs replaced the more numerous farms with fewer animals. This trend has only increased over time resulting in 7 650 farms nationwide reporting hogs with approximately 1 814 hogs per farm (Statistics Canada 2020b). This is a decrease from the 452 935 farms reporting pigs in 1921 (Brisson 2014). Of Canadian swine operations in 2011, 41.4% were finishing operations, bringing pigs up to market weight, followed by farrow-to-finish and nursery operations, 29.4% and 29.2% respectively (Brisson 2014). Farrow-to-finish operations handle pigs for the entirety of their life, from the breeding of sows, feeding, to finishing and selling. Nursery operations grow pigs up before selling to market or finishing operations which grow the pig up to market weight. Largely driven by the increase in technology and a drive for more efficiency, this rapid industrialization has allowed for greater profits for farms incorporating these modifications (Beaulieu, Bédard and Lanciault 2001). This change in farming culture to intensive farming and concentrated animal feeding operations can lead to problems of their own. The larger populations of animals in closer proximity can increase the possibility of emerging infectious diseases and exacerbate outbreaks (Hollenbeck 2016). These farming operations may act as reservoirs for antimicrobial resistance as well, which can reduce the efficacy of treatments when diseases occur (Alvarado et al. 2009). In 2014, antimicrobials were used more in agriculture than in human applications (Ontario Ministry of Agriculture 2016). Canada discourages the use of antimicrobials for use as growth promoters (Government of Canada 2018).

Diseases in the Canadian swine industry cause direct damages to the industry through treatment costs, losses of livestock, and reduced growth or production rates. Between 2004 and 2009, porcine circovirus (PCV) associated diseases were estimated to have cost the

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Canadian swine industry around $560 million (Agriculture and Agri-Food Canada 2015). Porcine reproductive and respiratory syndrome virus (PRRSV) was previously estimated to result in $130 million per year in losses to industry in Canada (Canadian Food Inspection Agency 2018; Mussell et al. 2011). Porcine epidemic diarrhea virus (PEDV) was estimated to cost up to $432 per sow depending on management practices (Canadian Food Inspection Agency 2018; Engele and Whittington 2014). Indirect costs of diseases include reducing faith in the quality of Canadian swine products, such as live exports, and reduce the demand for Canadian swine exports to other countries. An example of this indirect cost is when the price of goods was driven up due to concerns of the Canadian swine market arising from the PEDV outbreaks in the United States and Canada in 2012 (Statistics Canada 2014). Other concern-worthy pathogens include strains of Escherichia coli that are associated with post-weaning diarrhea (Public Health Agency of Canada 2012). For example, in Ontario it was found that pig herds with K88 E. coli post-weaning diarrhea had reduced growth rates and higher mortality (7% as opposed to 2%) than uninfected farms, which was calculated to theoretically cost a 500-sow operation $20 000 (Amezcua et al. 2002). During a study between 2001 and 2010 in Ontario it was found that 56% of gastrointestinal disease pathogens in piglets were

E. coli, Clostridium perfringens, C. difficile, or rotavirus (Chan et al. 2013).

Animal Disease Prevention Measures and Building Management

In order to reduce the incidence of disease and improve the health of the animals, national guidelines were developed to promote the well-being of animals by highlighting the requirements for animal care, such as the description of housing facilities (National Farm Animal Care Council 2014). The recommendations for the housing environment can promote the healthy growth of animals, which is also ideal for the operators financially (Choinière and Munroe 1993; National Farm Animal Care Council 2014). Among the recommendations and requirements are ventilation, temperature, and air quality, three interrelated and topical factors for this body of research (National Farm Animal Care Council 2014). Temperature is dependent on the age and size of animals varying between 10°C and 38°C, where younger piglets require higher temperatures (National Farm Animal Care Council 2014). Ventilation must be appropriate to maintain clean air without causing excessive disruptions linked to increases in ammonia (National Farm Animal Care Council 2014). Good air quality includes

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considerations for ammonia, dusts, and microorganisms (National Farm Animal Care Council 2014). While ammonia has recommended concentration limitations, <25 ppm, airborne microorganisms have no such recommendations provided (National Farm Animal Care Council 2014). Ventilation systems used in Canada tend to be mechanical, natural, or a hybrid system (Pouliot, Ricard and Dufour 2011). Hybrid systems are a combination of natural and mechanical ventilation systems. Natural systems use building features, such as vents, to modify the flow of air depending on the temperature required, decrease temperature during warmer seasons and increase the temperature during cold seasons (Pouliot, Ricard and Dufour 2011). Mechanical ventilation systems use fans to draw air in or out, i.e. positive and negative-pressure systems, respectively (Pouliot, Ricard and Dufour 2011). The most utilized mechanical ventilation system is the negative-pressure systems (Pouliot, Ricard and Dufour 2011) (Figure 0-1).

Figure 0-1 Examples of A) positive and B) negative-pressure ventilation systems. Blue arrows indicate the movement of air within the buildings. Modified figure from Pouliot, Ricard, and Dufour 2011.

An issue that can be encountered in negative-pressure systems is the draw of new contaminants into the building through leaks, although methods such as controlling the exhaust fans and insulation can mitigate this (Pouliot, Ricard and Dufour 2011). The European Union has established limits on the emissions of ammonia from swine buildings depending on the type (weaning, farrowing, fattening, or gestating) (Santonja et al. 2017). However, unlike ammonia, there is no established limitations or guidelines set on the biological content (bioaerosols) emitted from swine buildings and there is a lack of control via biosecurity measures.

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Biosecurity in Swine Barns

Biosecurity of swine buildings in Canada emphasize three strategies: reducing introductions of disease into herds, minimizing the spread between animals of the herd, and preventing the release of pathogens from the infected herd to another uninfected swine herd (Technical Committee on Biosecurity 2010). These are summarized as the terms bio-exclusion, bio-management, and bio-containment, respectively (Technical Committee on Biosecurity 2010). The Canadian Swine Health Board (CSHB) developed a standard composed of best management practices for biosecurity (Technical Committee on Biosecurity 2010). The standard describes farm activities and associates them with possible modes of pathogen transmission (e.g., direct or indirect contact). Biosecurity practices are then recommended and the principles of each practice explained. Among the mentioned biosecurity practices, it is important to highlight the differences between bio-management, bio-containment, and quarantine. Bio-management and bio-containment represent the set of procedures used to prevent the further dissemination either within or between swine herds with a pathogen already present (Technical Committee on Biosecurity 2010). A quarantine on the other hand refers to the protocols that handle the isolation of incoming animals from separate populations of known or unknown health status (Lipman and Leary 2015). Segregation aims to maintain distances between animals of unknown health status or known infected to ensure healthy animals are not exposed. Sanitation refers to the cleaning or disinfecting of areas housing animals. Finally, flow management controls the movement of animals to prevent infected or possibly infected animals from being moved together or to herds of healthy animals (Technical Committee on Biosecurity 2010). In the standard, biosecurity practices to prevent airborne transmission (bioaerosols), an indirect mode of pathogen transmission, are only split into segregation and flow management. In Canada, although there is concern regarding the potential transmission of disease through the air, there is no explicit requirements to mitigate bioaerosols aside through distancing animals from potential exposure (Canadian Food Inspection Agency 2012; Technical Committee on Biosecurity 2010). This may leave the industry susceptible to diseases transmitted by bioaerosols.

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Swine Barns’ Bioaerosols and Their Behaviour

Bioaerosols are aerosolized organic particles composed of intact, fragments, or products of organisms such as bacteria, fungi, and viruses, and characterized by particles with an aerodynamic diameter of a few nanometer up to 200 µm. Objects, such as particles, travelling through a medium, such as falling through the air, undergo multiple types of forces, like the forces of gravity and drag. Particles have what is called a Reynolds number calculated from characteristics of the gas they travel in and the particle itself, size, and velocity. When the Reynolds number is small (Re ≪ 1), typical of small particles with low velocities, Newtonian calculations are replaced with Stoke’s Law. This then describes the physics of small particles in a medium. The aerodynamic diameter of a particle is the equivalent diameter of a sphere with a density of 1 g/cm3_{(density of water) and the settling velocity is}

the same as the particle. The aerodynamic diameter of the aerosolized organic particles greatly influences the distance bioaerosols can travel and the time spent in the air. For example, particles greater than approximately 100 μm may only travel less than 1 meter before being deposited while smaller particles can travel much further (Lighthart et al. 1991). Particles were aerosolized into an vertical air flow of approximately 0.0233 m/s, identified as quasi-laminar flow (Lighthart et al. 1991). Furthermore, the health impact of bioaerosols can change based on the aerodynamic diameter of the particles as this influences where in the respiratory tract the particle can deposit, smaller particles reaching deeper (such as into the lower respiratory tract) (Roy and Milton 2004; Thomas et al. 2008). In general, particles in swine buildings were found to have an MMAD of 10 – 19 μm according to cascade impactors (Maghirang et al. 1997; O’Shaughnessy, Achutan and Karsten 2002). Microorganisms are often contained in or on particles often composed of a mixture of microbes and other organic components (e.g., proteins, structural phospholipids). Once aerosolized, microbial viability or infectious potential (for viruses) is greatly impacted by environmental stresses such as temperature, relative humidity, presence of chemical compounds, gases (e.g., ozone), particle size, radiation (e.g., ultraviolet), and water activity (Haddrell and Thomas 2017). The effect of these environmental conditions varies by microorganisms present with virtually no universal rule regarding the most detrimental conditions. Temperature has an effect on microorganisms whether in airborne particles or not (Hermann et al. 2007; Verreault et al. 2015). Relative humidity has an important role in

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microorganism survival in the air (Hatch and Dimmick 1966; Liu et al. 2012). Frequently, viruses exhibit differences in infectivity under changing relative humidity conditions (Hermann et al. 2007; Verreault et al. 2015). No rule on the effects of relative humidity on viruses can be established as viruses each react differently, including some viruses that have enhanced survivals at low relative humidity. (Turgeon et al. 2016). However, utilizing the general susceptibility of organisms to some chemical effects, such ozone, can be implemented to disinfect the air (Tseng and Li 2006). The efficiency of ozone to inactive microorganisms can vary by concentrations and the type of microorganism (Tseng and Li 2006). Ultraviolet (UV) radiation can have detrimental effects on airborne microorganisms (Cutler et al. 2012). This, similar to ozone, provides an opportunity for using UV for treating air (Cutler et al. 2012). Particle size can provide some protective effects for microorganisms inside but is associated with the time particles remain suspended in the air, which affects the distance particles can travel (Haddrell and Thomas 2017).

Swine building bioaerosols have been characterized throughout the years to establish pathogen aerosolization and compositions. Normal activities by swine, such as breathing, moving, and eating can generate bioaerosols. Greater numbers of pigs and larger pigs have been associated with greater airborne microbial concentrations (Rodríguez de Evgrafov et al. 2013). Total microorganisms in the air of swine buildings determined by cell staining found a range of 105_{to 10}7_cells/m3 _{(Chi and Li 2005; Rodríguez de Evgrafov et al. 2013).}

Culturable airborne bacteria in swine buildings are in the range of 105_{to 10}6_{CFU (colony}

forming units)/m3 according to previous studies (Chi and Li 2005; Cormier et al. 1990; Duchaine, Grimard and Cormier 2000). Bacterial 16S rRNA gene copies in swine air samples in Quebec was quantified between 108 and 109 copies/m3, a greater concentration than culturable quantifications (Bonifait et al. 2014; Nehme et al. 2008). Airborne fungal quantities ranged from 102 to 103 CFU/m3 in swine buildings (Chi and Li 2005; Duchaine, Grimard and Cormier 2000). As described, bacteria are more numerous in swine buildings than fungi although the quantities of total viruses cannot be generalized as there is no universal infectious or genomic virus quantification methods available as is the case for bacteria (16S rRNA gene).

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Analyses of the bacterial communities in the air of swine buildings found predominantly Bacteroidetes and Firmicutes (Kumari and Choi 2014; Nehme et al. 2008; Rodríguez de Evgrafov et al. 2013). Diversity analyses of archaea present in swine air has previously shown a community dominated by Methanosphaera stadtmanae (Nehmé et al. 2009). Streptococcus suis was detected in swine building air in concentrations up to 106 S.

suis gene copies/m3, other Streptococcus species were also detected by sequencing (Bonifait et al. 2014; Nehme et al. 2008). Many species of Enterococcus have been detected using isolation and biochemical assays of swine air samples, including E. faecalis (Chapin et al. 2004). In Quebec swine building air, zoonotic pathogens Staphylococcus aureus (and methicillin resistant strain), Campylobacter, Clostridium species, Enterococcus species, E.

coli, Salmonella species, Mycobacterium avium, Listeria monocytogenes, and Yersinia enterocolitica were detected by qPCR and cultures (Létourneau et al. 2010; Pilote et al.

2019). Another bacterial species detected in the air is Mycoplasma hyopneumoniae (Dee et al. 2009; Fano, Pijoan and Dee 2005). Viruses that were detected in swine building air are PRRSV, PEDV, IAV (influenza A virus), and porcine circovirus (PCV2) (Anderson et al. 2017). Seasons influence the community composition of airborne bacterial populations in swine buildings as increases in Proteobacteria and Actinobacteria could be observed during the summer (Kumari and Choi 2014). Season has previously been shown to have an impact on the total bacteria in the air, as quantified by qPCR of 16S rRNA copies, resulting in more bacteria in the air in the winter than in the summer, likely an effect of ventilation fans moving air and bioaerosols out of the building in the summer (Nehme et al. 2008). Additionally, the type of manure treatment system employed at a facility has an impact on the airborne bacterial communities (Kumari and Choi 2015). This is in line with previous findings that particles in the swine building air were derived from feces as well as skin and feed (Cambra-López et al. 2010). The origin of the particles in swine buildings can vary depending on the size of the particle (Cambra-López et al. 2010).

Size of airborne particles can have an impact on the distance the particle can travel but bacteria and viruses are not necessarily present in one size fraction or uniformly present across size fractions (Alonso et al. 2015). Microbial content of bioaerosols has little impact on particle size, as such viruses and bacteria can travel on particles of larger size and do not travel individually but in aggregates (Verreault, Moineau and Duchaine 2008). Viruses such

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as IAV, PEDV, and PRRSV were found on varying particle sizes up to 10 μm at differing concentrations (Alonso et al. 2015). Airborne culturable bacteria were found to be in greater concentrations on particles larger than 8 μm than smaller (Gibbs et al. 2004). Fungi were similarly found in greater concentrations for cultures in larger particles (Gibbs et al. 2004). Endotoxins were in greater concentrations in particles larger than 4.7 μm (Siggers et al. 2011). Similarly, culturable enteric bacteria were more numerous on larger particles, especially >7 μm (Siggers et al. 2011). Studies of bacterial transmission by air in swine buildings has found fairly limited distances possible for most common bacteria. Inside buildings, indirect transmission was found by Escherichia coli at short distances of up to 20 ft (~6 m) away (Cornick and VuKhac 2008). Culturable bacteria were detectable up to 150 m downwind from a swine building at concentrations more than twice as great as was detectable upwind (Green et al. 2006). Certain bacteria, such as Mycoplasma, were detected at long distances, 9.2 km, from their origin (Otake et al. 2010). Viruses can be detected at long distances from their sources as well such as PEDV that was detected up to 10 miles (~16.1 km) from a source (Alonso et al. 2014). Another example is the detection of infectious PRRSV by bioassay in samples up 9.1 km from the source (Otake et al. 2010).

Sampling of Swine Buildings Associated Bioaerosols (Principles and

Limitations)

Strategies for sampling aerosolized organic particles should take into consideration temporal and spatial variations and the concentration of bioaerosols inside swine buildings. In general, bioaerosols concentrations in swine buildings are known to be affected by the activity of animals, location of the sampler in the rooms, and seasons as examples (Jerez, Zhang and Wang 2011; Kim et al. 2008; O’Shaughnessy, Achutan and Karsten 2002). In addition to changing the concentrations of bioaerosols, the composition (such as microorganism community abundances) are affected by many factors. The effect of season on concentrations and composition of bioaerosols should encourage extended time frames for studies sampling the air of swine buildings (Kumari and Choi 2014; Nehme et al. 2008; Thorne, Ansley and Perry 2009). The influence on bioaerosols composition by swine feces and proven effect of manure treatment indicates that during sampling the manure handling should be identified for better comparisons with studies or sites (Kumari and Choi 2015;

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Letourneau et al. 2009). In addition, due to the effects of the types of animals being raised, age and weight, the type of operations needs to be reported for improved cross-comparisons (Yan et al. 2019). Due to the high particulate loads in the air in swine buildings, it is important to choose samplers that can handle the high loads without clogging or significantly losing efficiency (Predicala et al. 2002; Thorne et al. 1992).

Bioaerosols Monitoring and Sampling

The determination of aerosolized particle concentrations and size is important due to the health risks that are associated with total airborne particles as well as specific size fractions (for example particulate matter (PM) 2.5) (Kappos et al. 2004; Polichetti et al. 2009; Roy and Milton 2004). Particulate matter (PM) is a terminology for identifying particulate size fractions based on maximum size, such as PM2.5 indicates particles with an aerodynamic diameter of less than 2.5 μm (Environment Canada and Health Canada 2000).

The Optical Particle Sizer 3330 (OPS) manufactured by TSI Incorporated (TSI Incorporated, Minnesota, USA) actively draws the air particles into the machine to be monitored. Counting and sizing are performed using a laser which when it interacts with aerosol particles generates a light pulse that a detector records the intensity of to provide information on the size. The OPS can size particles between 0.3 and 10 μm over 16 channels and operates at a flow rate of 1 L/minute. The OPS has a maximum reading capacity of 3000 particles/cm3. For environments with particle concentrations that could exceed this limit, diluters are available from TSI at rates of 100 to 1 and 10 to 1 (Aerosol Diluter Model 3332, TSI Incorporated).

Analyses of bioaerosols require sampling or collecting air samples. There are liquid based air samplers (e.g. impingers and wet cyclones) and air samplers using dry filters as collection substrates. Liquid air samplers are ideal airborne microbial quantification with culture-based methods.

All glass impingers (AGI, Ace Glass Impinger-30, BGI Instrument Inc., Butler, NJ, USA) are liquid-based samplers that use high flow rates to collect air particles into a solution (e.g. phosphate buffered solution). The distance between the inlet nozzle submerged into the collection substrate and the bottom of the collection recipient is typically denoted in the

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name, such as AGI-30 (nozzle located 30 mm from the bottom). The AGI-30 is rated for a flow rate of 12.5 L/min. The D50 representing the aerodynamic diameter for a 50% particle population capture, for the AGI-30 has been reported at 0.3 μm (Jensen and Schafer 1998; Nevalainen et al. 1992). The bend in the inlet gives the sampler a shape similar of that found in the human respiratory tract but can come at the cost of sampling efficiency for particles being larger than 5μm, with reduction down to 20 to 30% for particles larger than 10 μm (AGI-30 cut-off diameter) (Springorum, Clauß and Hartung 2011; Willeke et al. 1992).

Another liquid-based air sampler is the wet cyclone Coriolisμ Biological Air Sampler (Bertin Technologies, Montigny-leBretonneux, France). The Coriolisμ has an adjustable flow rate between 100 and 300 L/min. Samples are collected in approximately 15 ml of solution. The Coriolisμ has a D50 of 0.5 μm. The operation is based on the formation of a cyclone in the collection recipient which impacts particles into the swirling solution and the wall of the recipient. The Coriolis Biological Air Sampler has previously been used in swine buildings for culturable bacteria and biological molecular analyses (Bonifait et al. 2014; Naide et al. 2018). In re-aerosolization experiments, it was reported that the Coriolisμ resulted in less re-aerosolized bacteria than an liquid impingement type sampler and led to an increase in bacterial concentrations in the collection liquid (Lemieux et al. 2019). Although this introduced a new bias, a concentrating factor, this can be compensated for by bringing the volume of liquid back to the original quantity (Lemieux et al. 2019). Collection type, liquid or filter, can affect the proportion of bacterial and fungal communities detected using HTAS (high-throughput amplicon sequencing) methods (Mbareche et al. 2018a). This extends even to samplers utilizing the same collection media, such as the difference in community compositions reported between a Coriolisμ and SASS2300, both collect air samples in liquid solutions (Mbareche et al. 2018a).

The Electrical Low-Pressure Impactor (ELPI) (Dekati Ltd, Kangasala, Finland) collects and counts particles distributed into 15 size fractions from 6 nm to 10 μm at a flow rate of 10 L/min. The ELPI combines cascade particle impaction (collection), electrical detection (counting), and low pressure sampling (Keskinen, Pietarinen and Lehtimäki 1992). The cascade impactor is composed of 14 collecting stages with decreasing D50 as air is accelerated and drawn through the impactor. For particle counting, the particles initially pass

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through a unipolar charging field generated from a corona charger to charge the particles (Keskinen, Pietarinen and Lehtimäki 1992). A unipolar charge is known to have a greater efficiency for charging particles over bipolar (Intra and Tippayawong 2011). When the particles are distributed on the stages the electric current is passed and measured (Keskinen, Pietarinen and Lehtimäki 1992). The low pressure environment is essential for the efficient sampling of nano-sized particles (Keskinen, Pietarinen and Lehtimäki 1992). A comparison of the ELPI to another particle sizer, Scanning Mobility Particle Sizer (SMPS), found the ELPI to be similar in efficiencies (Marjamäki et al. 2000). There has been some success in the collecting and counting of bioaerosols using the ELPI (Xu et al. 2013).

Mitigating Bioaerosols in Swine Confinement Buildings

The risk of bioaerosols carrying pathogenic agents from contaminated to naïve herds necessitates the consideration of air treatment technologies to reduce animal exposure to pathogens. This has led to the evaluation of commonly used technologies as well as those primarily designed for treatment of other emissions, like ammonia, for their co-removal of bioaerosols.

Widely-used and commercially available mechanical filtration has been proposed and evaluated in-field for use in the swine industry because of its proven efficiency for removing airborne particles in other environments. Mechanical filtration is the process of physically removing particles from the air using mechanism such as impaction, diffusion, or straining (Galka and Saxena 2009). The filters can be composed of different materials affecting their efficiencies and sizes, one example is microglass (Galka and Saxena 2009). Filtered air was able to reduce the number of infected pigs when using mechanical filters in ducts connecting to PRRSV infected pigs to naïve pigs (Dee et al. 2005). The evaluated pre-filters and filters had European filter grading of EU8 and EU13 (Dee et al. 2005). The EU grades correspond to efficiencies of filters to remove particles for filters graded for use in Europe, while ASHRAE has developed the minimum efficiency reporting value (MERV) for America. The EU8 corresponds to a MERV14 that removes <75% of particles 0.3 to 1 μm. EU13 is equivalent to a MERV19, both of which are considered HEPA filters capable of removing >99.97% of particles >0.3 μm. Various other mechanical filter prototypes were challenged using a droplet aerosol generator from liquid solutions of PRRSV, equine arteritis virus

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(EAV), bovine enterovirus 1 (BEV), Actinobacillus pleuropneumoniae (APP), and S. aureus (Wenke et al. 2017). The study examined 4 prototype filters: a polyester prefilter (MERV6-8) with glass fiber filter (MERV16), a synthetic organic prefilter (MERV6-(MERV6-8) with glass fiber (MERV16), a polyester filter (MERV9-13), and a glass fiber and wool filter (MERV14-16) (Wenke et al. 2017). BEV, APP, EAV, and S. aureus were best removed by the glass fiber and wool filter, while PRRSV was best reduced by the polyester prefilter and glass fiber filter combination (Wenke et al. 2017). Filtration of barns, using MERV 14 and MERV 16, showed improved protective effects for herds against both Mycoplasma hyopneumoniae and PRRSV (Dee, Otake and Deen 2010). This study also proved the potential use of antimicrobial filters which similarly protected animals from pathogens (Dee, Otake and Deen 2010).

Dee and collaborators provide evidence of protection against incoming pathogens for negative-pressure buildings using mechanical and antimicrobial filters, a bio-exclusion system (Dee, Otake and Deen 2010). The other potential use of air treatment technology is on the exhaust air of buildings, bio-confinement system. The use of mechanical filtration for exhaust air can be challenging as the high particulate load in the air can cause premature clogging and reduce the efficiency of the filter. In order to compensate for this, pre-filters with a lower capture efficiency against airborne particles may be installed to increase the lifespan of the more efficient and expensive filter (>3 years). Such combinations of filters was evaluated on the field in previous studies or recommended by organizations (Dee, Otake and Deen 2010; Janni et al. 2018; Pouliot, Ricard and Dufour 2011). However, pre-filters will still require regular replacement (up to a year). This drives the interest in alternative air treatment technologies that do not suffer from clogging issues, such as antimicrobial filters which are less efficient mechanical filters composed of fibers embedded with bactericidal and virucidal agents. Antimicrobial filters inactivate airborne microbes without retaining particles, which improves the filters lifetime. Experiments during which PRRSV were nebulized from a suspension and passed through an antimicrobial filter into rooms containing pigs resulted in no infections in animals, in contrast the animals became positive for PRRSV in the absence of an antimicrobial filter (Batista, Pouliot and Urizar 2010). This shows that the antimicrobial filters can reduce the infectious potential of PRRSV and could be speculated to be added to the exhaust fans to reduce the emitted viruses.

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Technology that has been implemented for the reduction of odors and ammonia, such as to comply with European standards, have been investigated as being multipurpose for the reduction of bioaerosols. Air scrubbers are air treatment systems that either use water or acids to capture and solubilize airborne compounds (Figure 0-2). Water-based scrubbers utilize a spray of a recirculating solution over a bed facilitating the movement of ammonia into a liquid phase (Van der Heyden, Demeyer and Volcke 2015). Acid, typically sulfuric acid, scrubbers decrease the pH changing the environment to prefer ammonium over ammonia which then produces ammonium sulfate salts (Van der Heyden, Demeyer and Volcke 2015). In either case, the washing liquid is recycled until ammonium levels are too high to efficiently continue reducing airborne ammonia concentrations and requires the removal and replacement of the spraying solutions (Van der Heyden, Demeyer and Volcke 2015). A sulfuric acid scrubber applied to the exhaust air of a swine building resulted in a reduction of culturable bacteria by 70% (Aarnink et al. 2011). Tests on small-scale air scrubbers using peracetic acid in laboratory conditions reduced nebulized Enterococcus faecalis at an average of 100% (Aarnink et al. 2011).

Figure 0-2 Diagram of air scrubber systems including acid and biological scrubbers. A) A packing material. B) A recirculated acid, water, or nutritive solution with fresh solution available.

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Another air treatment technology is the use of biofilters, relying on microorganisms instead of chemical or physical capture of contaminants compared to air scrubbers. Biofilters use a porous synthetic media as a bed to house microbial communities (biofilms) to metabolize airborne contaminants of concern passing through the biofilter. The media can be composed of different components or mixtures of them, such as wood chips, compost, and corn cobs (Akdeniz, Janni and Hetchler 2016; Chen et al. 2009; Nicolai and Janni 2001). Biofilters composed of wood chips were able to reduce airborne methicillin-resistant

Staphylococcus aureus (MRSA) up to 95% depending on the type of wood chips when

connected to exhaust air of a swine building, farrow to nursery operation (Ferguson et al. 2015). Air from a fattening building was treated using five biofilters composed of different bed materials and analyzed for culturable bacteria and fungi reduction (Martens et al. 2001). The results varied by filter bed type and target but was able to decrease culturable bacteria up to 95% (Martens et al. 2001). Percolating biofilters, or bio-trickling filters, utilize the same mechanism of air scrubbers with liquid spraying over a bed to move contaminants from the air into the liquid phase except a biofilm of microorganisms, like in biofilters, metabolize contaminants, such as oxidizing ammonium (Van der Heyden, Demeyer and Volcke 2015). These air treatment systems have advantages over mechanical filtration for avoiding clogging issues and can treat large volumes of air. In all types of air scrubbers the removal of used water must be performed over time as the salts build-up in the recirculated water although for differing reasons, toxicity to microorganisms in biofilms or to prevent clogging in acid scrubbers (Van der Heyden, Demeyer and Volcke 2015). A majority of studies have investigated the use of percolating biofilters for their ability to reduce gases and odours from the air of swine buildings with few investigating the potential biosecurity applications (Belzile et al. 2010; Melse, Ploegaert and Ogink 2012; Van der Heyden et al. 2019). While in small-scale experiments percolating biofilters showed a 10x reduction of bacteria, Aarnink and collaborators found a 279% increase in bacteria downwind of the percolating biofilter (Aarnink et al. 2011; Lévesque et al. 2017). The concern with potential release of microorganisms from percolating biofilters can include pathogens such as Legionella which has been detected downwind of percolating biofilters in increased concentrations (Walser et al. 2017). Using molecular and immunological methods of detection, Legionella spp and

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et al. 2017). The engineered environment of percolating biofilters, a reservoir of water then sprayed, is a prime environment for Legionella and should be examined in further studies (Walser et al. 2017).

Inside swine buildings the physical capture of particles in the air is an important aspect of the removal of airborne contaminants including bioaerosols. One such mechanism employed to reduce the airborne contaminants inside the buildings is oil sprinkling (Paszek et al. 1998). Oil sprinkling has been shown to reduce airborne dust in swine buildings, possibly up to 86% (Lévesque et al. 2018; Siggers et al. 2011). While not effective for bacteria or fungi in the air, it was effective (82.5%) for reducing airborne endotoxins (Siggers et al. 2011). Oil sprinkling treatment was more effective at removing larger particles although the well reduced endotoxins were on smaller particles (Siggers et al. 2011). Bacteria in the air of rooms with sprinkling were less on larger particles but greater or equal on smaller particles in the control rooms (Siggers et al. 2011). However, in a study comparing the effects of different solutions spraying in buildings of grower pigs found that a soybean oil sprayed over 24 hours was able to reduce the airborne bacteria compared to just water (Kim et al. 2006).

Ozone is known to have bactericidal effects, including when the ozone was aerosolized and the bacteria was exposed on surfaces (Kowalski, Bahnfleth and Whittam 1998). Ozone had been shown to reduce eukaryotic viruses and phages in water (Shin and Sobsey 2003). In indoor environments, ozone has been proposed for reducing the risk of exposure of people to bioaerosols (Huang, Lee and Tai 2012). Aerosolized phages were found to be inactivated by ozone, particularly at higher relative humidity (Dubuis et al. 2020; Tseng and Li 2006). Relative humidity is important as there is a relation between water and ozone, an interaction likely generating toxic oxygen radicals (Foarde, Vanosdell and Steiber 1997; Li and Wang 2003). A relationship between the complexity of the capsid structure and ozone susceptibility was previously reported (Tseng and Li 2006). An increasingly more complex capsid resulted in more resistant phages to ozone (Tseng and Li 2006). The only enveloped virus, Phi6, was the most sensitive although it had a greater capsid complexity than PhiX174 and as such the authors speculated that it was the envelope that caused the increased sensitivity (Tseng and Li 2006). This supports previous findings of f2 phage

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interactions with ozone that found the primary effect to be on the capsid proteins followed by a secondary effect on the nucleic acids (Kim, Gentile and Sproul 1980). This is argued in other studies and may suggest multiple confounding factors beyond capsid protein structures and nucleic acid such as type of nucleic acid (DNA, RNA, double-stranded, or single-stranded), the presence of an envelope, and thus being virus specific (Roy et al. 1981; Shin and Sobsey 2003; Tseng and Li 2006). This information was supported by the tests on four phages by Tseng and Li (2006), which found that although there were more capsid proteins for Phi6 over PhiX174, Phi6 was more sensitive to ozone and may have been due to the presence of an envelope (Tseng and Li 2006). PRRSV was also shown to be able to be inactivated on surfaces by ozone applied for 10 minutes (Yoon and Kim 2013). Ozone tested in swine buildings has resulted in either no effect on culturable airborne bacteria to an average of 30% reduction (at 0.03 ppm ozone applied), varying by study (Banhazi 2011; Elenbaas-Thomas et al. 2005). It is important to note the toxicity of ozone to animals as well, 0.1 ppm ozone applied in the air resulted in the loss of daily weight gain in pigs (Elenbaas-Thomas et al. 2005).

Electrostatic precipitation is the use of electrostatically charged particles and their removal from the air stream exploiting their charge. A corona discharge causes suspended particles in the air to gain a charge, positive or negative (Mizuno 2000). Through the combined use of additional electrodes, the charged particles can then be collected from the air column (Mizuno 2000). The electrostatic precipitation principle has been employed for the use of collecting bioaerosols by air samplers (Mainelis et al. 2002; Mainelis et al. 1999). Some electrostatic precipitator air samplers are able to match the BioSamplers, a liquid-based air sampler for the intention of bioaerosols capture, in efficiency utilizing solid growth media for collecting bacteria and maintaining their viability (Mainelis et al. 2002). Electrostatic precipitators have been evaluated for potential use in swine buildings for use against dusts (St. George and Feddes 1995). In a quarantine facility of a swine barn, an electrostatic particulate ionization system was employed and successfully reduced dust (64%) and bacteria (83%) (Pouliot et al. 2013). As implied earlier, this technology has also been applied for use in collecting and counting airborne particles. Through the combination of low pressures and electrostatic precipitation particles ranging from very small (5nm) to large (10μm) is possible using Dekati’s Electrical Low Pressure Impactor (ELPI). The ELPI

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functions through the production of a corona discharge and the counting and collecting of particles on aluminium foil substrates that a computer can detect the impact and register it.

Evaluating Efficiencies of Air Treatment Technologies

The various technologies implemented or studied for use in reducing bioaerosols of swine buildings can be evaluated using a variety of methods. This can include using artificial bioaerosols generated inside an environmentally controlled and dynamic aerosolization chamber (e.g. a laminar airflow in a wind tunnel) or naturally generated bioaerosols from operational swine buildings.

Artificial and natural bioaerosols

Generation of artificial bioaerosols can be useful for examining the survival of microorganisms in an aerosolization chamber under differing environmental conditions, such as relative humidity and temperature. Different technologies exist to aerosolize microorganisms. Broadly, microorganisms can be aerosolized from liquid solutions or as dry powders (Griffiths and DeCosemo 1994). Microorganisms in liquid solutions can be aerosolized through a variety of technology but all include a shearing mechanism of action (Griffiths and DeCosemo 1994). One jet-based technology is the Collison nebulizer, which can exist as multiple jet systems, such as the 6 and 24-jet systems. These systems generate aerosols from a liquid solution and when the solution is composed of microorganisms it can produce bioaerosols (May 1973). The particles produced are approximately 1-2 μm (May 1973; Peters et al. 1993). The Collison 6-jet has been used in experiments to determine the efficacy of disinfectants against aerosolized phages (Turgeon et al. 2016). The Collison has also been used to examine the airborne survival, movement in an experimental room, and reduction efficiency of ionization for PRRSV (Hermann et al. 2007; Hermann et al. 2006; La and Zhang 2019; La et al. 2019). Alternative nebulizer technology applied to swine pathogens was a mister, which may produce small particle sizes of 0.3 to 3 μm (Dee et al. 2006). Finally,

Streptococcus suis was previously nebulized in an aerosolization chamber using a bubbling

system to evaluate preferential aerosolization of the bacteria (Gauthier-Levesque et al. 2016).

The use of bioaerosols produced from nebulization of a solution or wet bioaerosols is common in bioaerosols studies due to the availability and standardization of protocols

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growing or amplifying microorganisms in liquid culture media. This may then lead to a lack of knowledge relating to the generation and fate of dry bioaerosols found in operational or commercial swine buildings. Some samples of settled dusts from swine buildings have already been used in aerosolization chambers and provided indigenous (natural) microbial populations (Thorne 1994). Grain dust was used as a substrate for microbial culture and was aerosolized as wet and dry bioaerosols to evaluate the properties of each (Thorne 1994). Greater concentrations of airborne bacteria and fungi were found following the generation of dry bioaerosols than for the wet (Thorne 1994). Particle sizes were also observed to be different between the productions of wet and dry bioaerosols, with dry bioaerosols being larger than the wet bioaerosols (Thorne 1994). Dry bioaerosols for aerosolization studies adds complexity because of the potential requirement to maintain viability or infectious potential or at least the ability to identify and quantify the studied microorganism (Hart et al. 2020). To this end, spore forming bacteria are ideal candidates for use because of their ability to survive harsh environments, such as lyophilization to generate powdered preparations of microorganisms (Hart et al. 2020). Hart et al. (2020) used Bacillus thuringiensis for experiments aerosolizing lyophilized bacterial spores, after other additive steps. The study by Hart et al. (2020) was recommended for general evaluations of bioaerosols and not specifically for swine buildings.

Standardized test dusts exist for the purpose of evaluating filters in a reproducible manner and calibrating measurement tools (O’Shaughnessy, Achutan and Karsten 2002). Standard test dusts are available in a variety of size distributions and are composed of dusts of known chemical composition and proportion (Powder Technology Inc., Burnsville, Minnesota). For example, the ISO 12103-1 A3 medium test dust (Powder Technology Inc.) is available with an MMAD of approximately 12 μm and is composed primarily of SiO2

(68-76%) (Personal communications). The 12 μm lies within the range of particles expected in swine buildings, 10 – 19 μm, and may then be a suitable model (Maghirang et al. 1997; O’Shaughnessy, Achutan and Karsten 2002).

Various dispersal mechanisms and standardized dusts are commercially available. The standardized dust allows for tests challenging filters using a consistent material. Dry powder aerosolization systems exist for a variety of reasons including the aerosolization of

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test dusts or for use in inhalable medicines, although the requirements for the dusts changes depending on intention (Chan 2006; Golshahi et al. 2011; Tiwari, Fields and Marr 2013). An issue that accompanies the aerosolization of powders is the natural clumping of particles with one another which requires considerations for dispersing the powders properly (Calvert, Ghadiri and Tweedie 2009; National Research Council 2006). The disperser type examples include nozzle, venturi, capillary tube, and fluidized beds (Calvert, Ghadiri and Tweedie 2009).

A venturi disperser called the Small-Scale Powder Disperser (SSPD) (TSI Inc, Minnesota, USA) can aspirate powder from a rotating plate. A venturi disperser uses shear forces generated from the suction of particles into high-speed air flows after a constricted section of tubing to disrupt the aggregates of the powder (Chen, Yeh and Fan 1995). The SSPD efficiency of dispersion is the size of the particles to be aerosolized, with losses in efficiency as particle sizes increases (Chen, Yeh and Fan 1995). A draw back to the SSPD is the limited sample possible for aerosolization as noted in the name, small-scale, requiring a small quantity of powder spread on the plate. Manual manipulation of the aspirator over the head can also lead it losses in aerosolization.

One such example of a powder disperser is the Solid Aerosol Generator (SAG) 410 (TOPAS-GmbH, Dreseden, Germany). The SAG 410 uses a nozzle-based disperser system which is fed powder by suction in controlled sample sizes using a belt and scrapper system. The SAG 410 has various models with differing flow rates, capacities, and properties, such as ranging potential outputs from 0.05 to 6000 g/h across all models. The standard SAG 410 can produce up to 500 g/h. The automated scrapper and belt system allows for consistent sample sizes to be aerosolized. SAG 410 and its models have been used in studies of air quality, cleaning, and filtration for generation of dusts to simulate real-world conditions (Bae et al. 2015; Bai et al. 2018; Blake, Wilson and Stewart 2018). Various filter materials were challenged using a SAG 410 nebulizing an A2 fine standard test dust (ISO Standard 12130-1) (Hasolli, Park and Rhee 2013). Another study evaluated borosilicate glass and electret HEPA filters using A3 medium test dust (ISO 12103-1) and A1 ultrafine test dust (ISO 12103-1) (Wang, Lin and Chen 2016). A SAG 410 was previously used to evaluate the removal of dust by technology intended for installation in subway trains (Bae et al. 2015).