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Article

Particulate Matter Concentrations and Fungal Aerosol in Horse Stables as Potential Causal Agents in Recurrent Airway Disease in Horses and Human Asthma and Allergies

by
Anna Lenart-Boroń
1,*,
Anna Bajor
2,
Marek Tischner
2,
Klaudia Kulik
1 and
Julia Kabacińska
3
1
Department of Microbiology and Biomonitoring, Faculty of Agriculture and Economics, University of Agriculture in Kraków, 30-059 Kraków, Poland
2
Department of Animal Reproduction, Anatomy and Genomics, Faculty of Animal Sciences, University of Agriculture in Kraków, 30-059 Kraków, Poland
3
University Centre of Veterinary Medicine UJ-UR, University of Agriculture in Kraków, 30-059 Kraków, Poland
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(18), 9375; https://0-doi-org.brum.beds.ac.uk/10.3390/app12189375
Submission received: 22 July 2022 / Revised: 13 September 2022 / Accepted: 14 September 2022 / Published: 19 September 2022
(This article belongs to the Special Issue Fungi Associated with Indoor Environments and Materials)
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This study provides a scientific basis to improve welfare of horses kept in stables, and to eliminate the causal agents of recurrent airway disease in horses.

Abstract

Exposure to bioaerosols associated with horse stable indoor environment and their health effects on people and horses has recently become of particular interest. Moreover, increasing frequency of recurrent airway disease (RAO) among horses made it necessary to search for the most probable causal agents of this disease and methods of their eradication. The study was conducted in two horse stables in southern Poland (Kraków and Tarnów). Particulate matter (PM2.5, PM4, and PM10) concentrations were determined photometrically, the concentration of fungal aerosol was determined by a six-stage impactor, and next generation sequencing (NGS) was used to determine fungal community composition in one of these stables. The highest PM concentrations were observed in Tarnów, but fungal aerosol levels were higher in the Kraków stable. Based on the NGS results, the three most prevalent fungal species were Wallemia sebi, Aspergillus penicillioides, and Epicoccum nigrum, all highly allergenic and potentially involved in the occurrence of RAO in horses. Spores of the detected fungi can penetrate deeply into the respiratory system. Therefore, this study suggests that examinations of particulate matter and fungal aerosol concentrations, along with species composition assessment, should be regularly conducted in horse stables.

1. Introduction

A large number of people worldwide are involved in the equine industry and there are more than six million leisure horseback riders in the EU, representing approximately 2% of the total EU population [1]. As a consequence, many people spend a considerable amount of time in stables, either as workers involved in taking care of and training horses, or as enthusiasts devoting their free time to horse riding [2]. As a result, both people and horses present in stables are exposed to airborne contaminants. Airborne contaminants associated with animal-dwelling environment include toxic gases, such as ammonia or hydrogen sulfide; inorganic particulates, such as soil dusts; non-viable organic particles, such as feed or feces droplets; as well as viable particulates such as bacteria and fungi, and their fragments and toxins. They are all referred to as bioaerosols [2].
Both occupational and non-occupational exposure to organic dusts and bioaerosols result in the occurrence of respiratory system and skin diseases, infections, toxic reactions, and syndromes associated with poor indoor air quality, e.g., sick building syndrome. The most common health effects can be encountered in places where particulate matter is associated with microorganisms or microbial particles which—as bioaerosol components—are transmitted by air–droplet or air–dust pathways and enter the body, e.g., through the respiratory tract [3]. Horse stables are the sites where such situations occur frequently. It has been reported that frequent contact with horses is associated with increased incidence of asthma, decreased pulmonary functions, and respiratory symptoms [4].
The growing interest in the horse business mentioned above is manifested in Poland by the change in the type of horse use and their increased status, as well as the change in the proportion between horse keeping for work and for recreational purposes. This is followed by attention paid to the conditions in which these animals are kept, including type and quality of buildings for horses or the quality of indoor air in horse stables [5]. The contamination of air inside animal housing is an important aspect of zoohygiene, and the concentration of organic dust, microorganisms, and their toxins influences animal welfare [6]. Even though much attention is paid to bioaerosol levels in livestock buildings, such studies are scarce in the case of horse stables. However, this issue needs thorough research for a number of reasons. Firstly, horse stables are indoor environments where people spend a lot of time for both occupational and recreational reasons [7]. Secondly, horses kept in stables are exposed to dust and bioaerosol particles, which have been suggested to be among the agents that contribute to airway and allergic diseases in horses, such as recurrent airway obstruction (RAO) [8].
Recurrent airway obstruction (RAO), commonly known as heaves or severe equine asthma, and inflammatory airway disease (IAD), are the most commonly recognized inflammatory diseases in horses [8]. They affect up to 20% of adult horses in the northern hemisphere [9]. In the case of disease exacerbation, horses develop a number of characteristic symptoms, including cough, nasal discharge, increased respiratory effort at rest, and exercise intolerance [10]. These diseases have been associated with hay feeding and stabling. The factors that trigger clinical signs of inflammatory airway diseases in horses include fungal spores, bacterial endotoxins, forage and storage mites, peptidoglycans, proteases, pollen, and plant debris, as well as inorganic particles [11]. Several fungal genera and species have been recognized as significant risk factors of RAO and IAD, but the majority of studies focus on the role of the genus Aspergillus, especially A. fumigatus in the pathogenesis of RAO and IAD in horses [11,12].
Concerns about indoor bioaerosol levels and exposure have increased over recent years. This is largely due to the recognition that exposure to bioaerosols is associated with a wide range of adverse health effects [13]. Specifically, inhalation of filamentous fungi-associated particles and dust may cause not only allergy, but also immunotoxic diseases or respiratory system diseases, such as asthma, infectious and invasive diseases, or may even have a carcinogenic effect [5]. Spending a lot of time indoors, particularly in rooms containing high particulate matter and bioaerosol concentrations, is a burden on the respiratory system of both animals and humans. It has been demonstrated that the occurrence of inflammatory airway conditions in horses are much more frequent when animals are stabled indoors [10,14]. According to the study by Nowakowicz-Dębek et al. [5], working in horse stables (e.g., as horse tenders) is classified as heavy due to a number of reasons, with the contamination of indoor air being among the most important factors contributing to the noxiousness of the work. Many studies suggested the major role of inhaling dust and fungal particles as being among the most important factors affecting the occurrence of inflammatory airway diseases and allergic reactions, both in humans and horses [5,6,10,11,13]. However, only some fungal genera (e.g., Aspergillus, Alternaria, Cladosporium, or Penicillium) have been reported as causal agents of allergies or inflammatory diseases [5,6,11]. For this reason, correct and reliable identification of the most prevalent fungal genera and species is crucial in understanding the role of airborne bioaerosols in the occurrence of airway diseases and allergies both in humans and in animals.
With this in mind, the specific aims of this study were (1) the examination of changes in the particulate matter (PM10, PM2.5, and PM1) levels, (2) culture-based assessment of fungal bioaerosol concentrations, and (3) next-generation sequencing (NGS) analysis to identify the airborne fungi in horse stables in southern Poland. The major drive for undertaking the study was the recurrent episodes of RAO and IAD, followed by the death of two horses in one of the horse stables in southern Poland. Therefore, in this study we aimed to find out whether the observed particulate matter and fungal aerosol concentrations, as well as the fungal species and genera identified by the NGS could contribute to the occurrence of respiratory diseases in horses, such as RAO, IAD, or respiratory allergies. Moreover, this study is an attempt to indicate whether the examined parameters pose a health risk (in the form of increased risk of allergies, occurrence of asthma or farmer’s lung) not only to animals, but also to people (workers, trainers, and horse enthusiast) who spend much of their time in horse stables.

2. Materials and Methods

2.1. Study Site and Design

The study was conducted in two horse stables in southern Poland: Kraków (Figure 1A) and Tarnów (Figure 1B). The samples were collected in two sites in each stable—indoors and outdoors. The measurements were carried out during one day, in stable weather conditions, without rain. Indoor measurements were conducted in four different times of day, varying in terms of the activities of horses and activities carried out inside the stables. These could have affected the air movement, particulate matter elevation, and increased concentration in the human and animal breathing zones. The focus was on the following times of day: early morning (no activity), feeding of horses in the morning, mid-day no activity (horses spent their time outdoors), and afternoon activity (e.g., horses coming back to the stable, cleaning activities, afternoon feeding). Outdoor measurement (outdoor background) was done once in each stable at noon. The total number of collected samples was 180 (i.e., 90 samples per each stable, including 18 samples outdoors and 72 samples indoors). In the Kraków stable, the measurements were conducted on 8 February 2022, while in the Tarnów stable they were collected on 15 February 2022.

2.2. Airborne Dust Concentration and Physical Parameter Measurements

Particulate matter concentration was measured using a LOOKO2 (ZAS Elżbieta Stochlińska, Węgrzce Wielkie, Poland) laser photometer with a built-in WiFi module, ensuring constant measurement and monitoring of dust particle concentration. After installation and configuration, the device was connected to the LOOKO2 server. The data were analyzed, calibrated, and then presented to the user. All LOOKO2 devices are calibrated before deployment and, immediately after configuration, join the global network of LOOKO2 sensors (https://looko2.com/heatmap.php (accessed on 24 August 2022). LOOKO2 has introduced a system that detects malfunctioning of smog sensors (caused by e.g., flooding with water), which prevents incorrect data on the air condition from being sent to the network. According to the manufacturer’s documentation, the sensor measures the concentrations of the following aerodynamic diameters of particles: 0.3–1 µm (PM1), 1.0–2.5 µm (PM2.5), and 2.5–10 µm (PM10) in 0.0001 m3 of air. The measurement resolution is 1 µm, and the maximum measurement error in the range of 0–100 µm is 10 µg/m3.
The measurement was conducted constantly throughout the day when the bioaerosol samples were collected. The device was placed at a height of 1.5 m above the ground to examine the air from the breathing zone of horses and humans. The data were measured once per minute, then logged once per 10 min, and the results are presented as means of six replicates from 1 h intervals.
The respirable fraction of particulate matter was assumed as smaller than 2.5 µm (i.e., PM2.5 and PM1). PM10 was assumed as inhalable dust. Temperature and relative humidity were measured using the Kestrel 400 Weather Meter (Nielsen-Kellerman, Boothwyn, PA, USA).

2.3. Fungal Aerosol Measurement

The concentration of fungal aerosol was measured using a 6-stage Andersen cascade impactor (WES-710 model, Westech Instruments, Essex, UK). It allows us to distinguish the following aerodynamic diameters of bioaerosol: 7 µm and above (stage one), 4.7–7 µm (stage two), 3.3–4.7 µm (stage three), 2.1–3.3 µm (stage four), 1.1–2.1 µm (stage five), and 0.65–1.1 µm (stage six). Particles smaller than 4.7 µm (i.e., stages three, four, five, and six) were considered respirable fraction (RF) [15]. Sampling time was from 1 to 3 min, depending on the preliminary assessment of air contamination based on the particulate matter concentrations. With the constant flow rate of 28.3 L/min, the examined air volume ranged from 84.9 to 141.5 L. The air sampler was placed at a height of 1.5 m above the ground to collect the air from the breathing zone of horses and humans. Fungi were incubated on Petri plates with Malt Extract Agar (MEA, Biomaxima, Lublin, Poland) at 30 °C for 4 days followed by another 4 days at 22 °C. The prolonged incubation of plates enables the growth of slowly growing strains at a lower temperature range [16]. After incubation, the colonies characteristic of fungi were counted and the results were presented as the number of colony forming units per one cubic meter of air (CFU/m3). The measurements were conducted in three replicates and the results are presented as means of these replicates.

2.4. Next Generation Sequencing

Next generation sequencing was performed on the DNA extracted from one HEPA filter (porosity of 0.3 µm), on which microorganisms were collected with a VKV125 Circular Duct Fan (R24Fans, Łódź, Poland) ventilator working over an 8 hour period. Mechanical lysis of the material was conducted in the FastPrep-24 homogenizer (MP Biomedicals, Irvine, CA, USA) with zircon balls. Fungal DNA was extracted using the Genomic Mini AX Bacteria + (A&A Biotechnology, Gdansk, Poland). The resulting DNA extract was purified using an Anti-Inhibitor Kit (A&A Biotechnology, Gdansk, Poland).
The DNA extract was used to prepare the amplicon library targeting the fungal ITS2 region (primers: ITS3F 5′-GCATCGATGAAGAACGCAGC-3′ and ITS4R 5′-TCCTCCGCTTATTGATATGC-3′ [17]). The library construction involved a two-step amplification using the Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2 (Agilent Technologies, Santa Clara, CA, USA) according to the ITS Metagenomic Sequencing Library Preparation protocol and a quality-check according to the Illumina qPCR Quantification Protocol Guide. Finally, amplicon libraries were sequenced (2 × 300 bp) using the Illumina MiSeq platform.
Sequencing data were processed via operational taxonomical unit (OTU) calling and taxonomic profiling using CLC Genomic Workbench (Version 12, Hilden, Germany) with Microbial Genomics Module Plugin (Version 4.1, Hilden, Germany). Sequences were classified using OTU clustering or picking (97% similarity, minimum score of 40) against the UNITE (Version 7.2) database. Data obtained on the occurrence of fungal species were used to calculate the relative abundance of the most common fungal genera in the examined filter sample.

2.5. Data Interpretation and Analysis

The observed concentrations of fungal aerosol were referred to the proposal of guidelines by the Team of Experts on Biological Factors (pol. ZECB) [18] on the recommended concentrations of airborne microorganisms by treating animal rooms as working premises contaminated with organic dust.
Statistical analysis was performed using Statistica v. 13 software (TIBCO, Palo Alto, CA, USA). The normality of data distribution was verified using the Shapiro–Wilk test. As the collected data were not normally distributed, further tests were based on non-parametric analyses. The significance of differences in the fungal aerosol and particulate matter concentration in different times of day as well as between indoor and outdoor measurement was determined with the Kruskal–Wallis analysis of variance. The significance of differences in the fungal aerosol and particulate matter concentration between the two stables was determined with the Mann–Whitney U test. Statistically significant relationships between the examined parameters (i.e., fungal aerosol concentration, its different aerodynamic diameters, particulate matter levels, temperature, and relative humidity) in the two examined stables were assessed using the Spearman’s correlation coefficient. All statistical analyses were performed at p < 0.05.

3. Results

3.1. Particulate Matter and Microclimatic Parameters

Particulate matter (PM) concentrations varied significantly both between the examined horse stables and between different times of day. They were much higher in the Tarnów stable than in the Kraków stable (Table 1) (p value ranged from 0.00025 for PM1 to 0.00028 for total dust). The differences between times of day were noticeable, but not as much as in the case of the two stables (the observed differences were not statistically significant at p < 0.05). In the Kraków stable, the lowest PM levels were observed during mid-day no activity (indoor background), while in the Tarnów stable, the lowest levels of PM were observed in the early morning.

3.2. Fungal Aerosol Concentration

Detailed values of fungal aerosol concentrations (CFU/m3) are presented in Table 2, while the percentage values of individual fractions of bioaerosol in the Kraków and Tarnów stables are shown in Figure 2 and Figure 3, respectively. As shown in Table 2, the highest concentrations of all fractions of fungal aerosol (and therefore total and respirable fraction) in the Kraków stable were observed during the feeding of horses. The observed concentrations during this activity were from 44 times higher than those observed during mid-day lack of activity (indoor background) for fraction F5 to even 344 times higher for fraction F2. The concentration of total fungal aerosol during feeding was 82 times higher than this recorded during mid-day, while respirable fraction was 68 times higher during feeding than during mid-day lack of activity. In the Tarnów horse stable, the highest concentrations of all fractions of fungal aerosol were observed during indoor activity (e.g., cleaning). However, the lowest concentrations were observed in the early morning, rather than in the case of indoor background. The highest concentrations of fungal aerosol were from 5 times higher from the lowest values (early morning) for fraction F1 to 32 times higher for fraction F6. What is also clear from Table 2 is that the fungal aerosol concentrations were much higher in the Kraków than in the Tarnów stable (differences statistically not significant at p < 0.05). These regularities are contrary to those observed in the case of particulate matter (i.e., much higher values recorded in Tarnów than in Kraków).
What can be seen in Figure 2 is that the share (%) of fine fractions of fungal aerosol (from 0.65 to 3.3 µm) predominate during different times of day, in both stables, but in varying proportions. In the Kraków stable, the share of the three smallest fungal aerosol fractions (i.e., from 0.65 to 3.3 µm) was the highest in the morning (84%), then dropped during feeding to 36%, then increased during the day to 51% (indoor background) and 75% during afternoon activities. What also needs to be noted is that the share of the three smallest fractions outdoors reached as much as 90%. In the Tarnów stable, on the other hand, the smallest share of the three finest fractions was observed in early morning (26%), then increased during feeding to 77%, but then dropped to 59% (mid-day, indoor background) and 53% during afternoon activities. The outdoor concentration of the finest fractions was 52%. Summarizing, the share of RF of fungal aerosol in the Kraków stable was as follows: 89.91% < 85.73% < 63.94% < 53.28% in the morning, during daily activities, during mid-day lack of activity, and during feeding, respectively. In the Tarnow stable, the share was: 84.97% < 70.21% < 69.15% < 55.95% during feeding, during mid-day lack of activity, during daily activities, and in the morning, respectively.

3.3. Taxonomic Composition of Fungal aerosol Components in the Kraków Stable

The fungal community was examined based on the next generation sequencing in the Kraków stable. The airborne fungi were classified into two phyla, i.e., Ascomycota and Basidiomycota, 21 classes, 45 orders, 77 families, 99 genera. As many as 103 taxa were identified at the species level. Figure 3A shows the 12 most abundant fungal taxa (11 identified to the species level), while Figure 3B shows the 10 most abundant genera identified in the examined filter sample, based on the NGS results. Supplementary Table S1 presents the list of 67 fungal species, the relative abundance of which exceeded 0.01%, and the number of reads were higher than 10. The genus Wallemia, with its species W. sebi, was the most abundant among the airborne fungi in the Kraków sample. The second most abundant species was Aspergillus penicillioides (18.92%, Figure 3A), and Aspergillus spp. was also found to be the second most abundant genus in the examined sample (30.68%, Figure 3B).

3.4. Correlation of Daily Activities in Stables with Bioaerosol Components

Changes in the total fungal aerosol along with inhalable particulate matter concentrations in different times of day, depending on the activity in the examined stables, are shown in Figure 4A,B for the Kraków and Tarnów stables, respectively. Similarly, changes in the respirable fractions of fungal aerosol and particulate matter throughout the day, are shown in Figure 5A,B for the Kraków and Tarnów stables, respectively. The lowest values of both total and respirable fungal aerosol, as well as inhalable particulate matter, were observed when there was no activity in both stables. In the Kraków stable it was indoor background (Figure 4A), while in the Tarnów stable it was early morning—before any activity began (Figure 4B). On the other hand, the highest concentrations of particulate matter and fungal aerosol correlated only in the Tarnów stable, where the highest values were observed during indoor activities. In the Kraków stable, the highest concentration of particulate matter was observed during indoor activities, while the highest fungal aerosol concentrations (both inhalable and respirable fractions) were observed during feeding of the horses.
Consequently, the correlations between the bioaerosol components and microclimatic parameters observed in the two stables are clearly different (Table 3). In the case of the Kraków stable, the components of fungal aerosol correlate positively with each other, which is not surprising. Temperature is a microclimatic parameter correlated with fungal aerosol (fraction F6, statistically significant positive correlation, 0.650 at p < 0.05). Temperature also correlated negatively with the particulate matter concentrations (statistically significant negative correlation values from −0.641 for PM1 to −0.896 for PM10). There was also a positive correlation observed between relative humidity and PM10 (0.786).
On the contrary, in the Tarnów stable, all fractions of fungal aerosol, as well as total fungal aerosol and its respirable fractions, correlated positively not only with each other, but also with all fractions of particulate matter and temperature. For example, there were very strong positive correlations between total and respirable fractions of fungal aerosol with temperature (0.960) or between total fungal aerosol and particulate matter fractions (ranging from 0.771 for PM10 to 0.804 for PM1) for respirable fraction of fungal aerosol. These correlations ranged from 0.703 for PM10 to 0.749 for PM1. Additionally, relative humidity correlated with fungal aerosol fractions, but this correlation was negative (i.e., −0.640 for total fungal aerosol, −0.827 for fraction F1).

4. Discussion

In this study, we applied a combination of methods to assess the quality of air in two horse stables in southern Poland (Kraków and Tarnów). The highest values of all PM fractions, as well as inhalable and respirable fraction of particulate matter, were observed during the activities carried out in the stables. Our observations are similar to those made by Elfman et al. [19], who also recorded considerable differences in the concentration of both fine and coarse fractions of particulate matter depending on the activities inside the examined horse stable. However, the values observed in our study are much lower than those reported by Elfman et al. [19], who observed the concentrations of organic dust within the ranges from 400 to 800 µg/m3, or those by Grzyb et al. [7], who observed the PM concentrations reaching 117–368 µg/m3 for PM10, 117–230 µg/m3 for PM2.5, and 107–225 µg/m3 for PM1. The PM data presented in this study were obtained from the optical LOOKO2 laser photometer. This type of device has now become very popular due to their lower cost compared to reference monitoring instruments [20]. However, what is important is that low-cost portable monitors can easily supplement existing networks of certified monitor stations and can be applied not only outdoors—their use is cautiously encouraged to monitor indoor air quality [21,22]. The low-cost sensors have been reported to have low accuracy, as the OM concentrations estimated by the light scattering sensors can be affected by environmental parameters. Among them, the relative humidity has been mentioned to affect the PM concentration due to varying hygroscopic properties of the particles that form the particulate matter fractions [20,23]. Even though a site-specific calibration, as well as the calibration under different humidity and temperature conditions, would improve the accuracy of the low-cost sensors [21], there are studies showing inter-model performance agreement in PM readings across 19 different PM sensors with no calibration equipment available or feasible [22]. Thus, the use of low-cost PM sensor devices in research of indoor air quality and comparing the obtained data obtained with the same equipment, calibrated before deployment, should be treated as reliable.
The concentration of fungal aerosol changed considerably depending on the time of day and the activities performed indoors in both stables. The highest concentrations of all fractions of fungal aerosol in the Kraków stable were observed during feeding. In the case of the Tarnów stable, the highest concentrations of all fungal aerosol fractions were observed during daily activities performed indoors, and similarly for the particulate matter. For this reason, statistically significant positive correlations between the PM and fungal aerosol levels were observed only in the Tarnów stable. Moreover, the concentrations of most fractions of fungal aerosol varied significantly between the times of day. The values of total and respirable fractions of fungal aerosol both in the Kraków and Tarnów stables are similar to the ones reported by Nowakowicz-Dębek et al. [5] or Witkowska et al. [6], who observed the total fungal aerosol concentrations within the ranges of 103–104 CFU/m3 and lower than the ones reported by Elfman et al. [24], who reported the total fungal aerosol concentrations exceeding 1 × 106 CFU/m3 in the Swedish stables. However, respirable fractions of fungal aerosol reported by Elfman et al. [24] were similar or somewhat higher to the results observed in our study. Due to the absence of regulations in Poland, the observed fungal aerosol concentrations were compared to the proposal of the Team of Experts in Biological Factors (Pol.: ZECB) [18] on the concentrations of airborne microorganisms, treating animal rooms as organic dust-contaminated working premises, which is 50,000 CFU/m3 for the total fraction and 25,000 CFU/m3 for the respirable fraction of fungi. In this respect, the concentrations of both total and respirable fraction of fungal aerosol exceeded the suggested values only once, i.e., during feeding of horses and only in the Kraków stable. According to Karwowska [25], the age and type of ventilation in the animal premises may be among the important factors affecting the concentrations of bioaerosol. In our study, both stables have a gravitational type of ventilation. The Kraków stable has two ventilation channels (0.4 × 0.4 m), eight windows per building, with an area of 300 m2 and 900 m3 cubature. The Tarnów stable has six ventilation channels of the same size and thirty-five windows per building with an area of 714 m2 and 2360 m3 cubature. In this respect, it can be assumed that both buildings are characterized by similar type and efficiency of ventilation.
Another important factor in terms of indoor bioaerosol levels is the share of respirable fraction. The greater share of respirable fraction in the total concentration of bioaerosol, the greater share of its particles can reach the lower respiratory tract of animals and people present in the stables [15]. According to Dutkiewicz [26], occupational exposure to small sized bioaerosols may cause not only infections related to the direct contact with microorganisms, but also may cause the diseases related to the mycotoxin and fungal glucan exposure. Among the consequences of respirable fraction of bioaerosol exposure are diseases such as bronchitis, obstructive pulmonary disease, allergic asthma, alveolitis, or organic dust toxic syndrome [15]. Brągoszewska et al. [27] observed the share of respirable fraction of bioaerosol exceeding 80% and suggested that this might contribute to the occurrence of not only allergic symptoms—respiratory tract diseases and such concentrations should be also treated as a disturbing observation. The percentage of respirable fraction observed in our study also suggests that the RF levels indoors might pose a risk to the animals and people staying indoors, as they have the potential to deposit in the tracheal, bronchial, or alveolar regions of the lungs [27].
We also attempted to find out whether there is a correlation between the fungal aerosol levels (and its fractions) and other parameters measured in this study, such as PM10, PM2.5, PM1, temperature, and relative humidity. Interestingly, we observed different regularities in the case of the two examined horse stables. The relationship between fungal aerosol fractions and particulate matter levels was more evident in the Tarnów stable than in Kraków, where fungal aerosol fractions seem not to be dependent on any of the other measured parameters. On the other hand, the changes in the fungal aerosol concentrations in Tarnów strictly follow the pattern of particulate matter and correlate significantly with all PM fractions, as well as with temperature (positive correlation) and relative humidity (negative correlation). These results differ from the ones obtained by Grzyb and Lenart-Boroń [28] in the Kraków Zoological garden, who observed a positive correlation between fungal aerosol concentrations and relative humidity of air. The aerosol particles in livestock premises (including horse stables) are mostly organic (above 90%) and may include, among others, water droplets, animal secretions such as saliva and feces, fragments of fodder, hair, straw, soil, and manure, as well as various types of microorganisms [29]. According to Islam et al. [30], exposure to the aerosol particles and its compounds from the livestock premises can cause death of animals and severe damage to the respiratory tract of people spending significant time inside the contaminated premises. However, the exposure level and thus the degree of hazard created by biological contaminants in indoor environments is controlled by a number of factors. Among these, the concentration, size distribution, microclimatic parameters of the indoor environment (including temperature and humidity), and ventilation are the most important ones [31]. Regarding the fact that there is no clear pattern of associations between microbial aerosol concentrations with particulate matter and microclimatic parameters that can be revealed neither from our studies, nor from the studies performed by other authors, other factors, such as particle size and species composition, need to be taken into account [18].
For this reason, the combination of classical and molecular identification approach was used in this study to determine the composition of the most prevalent fungal genera and species in the studied horse stables. The next generation sequencing-based assessment of fungal community composition, conducted at the Kraków stable, showed Wallemia sebi to be the most abundant among the fungal species, with relative abundance of 35.16%. These fungi are very well adapted to the environments characterized by low water activity. They can produce a number of bioactive metabolites, including tricyclic dihydroxysesquiterpenes: wallimidione, walleminone, walleminol, and azasteroids. There are rare reports on the involvement of Wallemia spp. in cutaneous and subcutaneous human infections, but the representatives of this genus are important allergens, as they produce massive amounts of tiny conidia (1.5 µm to 3.0 µm), ideal for aerial dispersal, and thus can easily enter the host lungs [32]. Moreover, conidia of W. sebi are covered with a layer of hydrophobins (small cell-wall proteins) which helps pathogens avoid detection by the immune system cells by masking them [32], making this fungus even more potentially dangerous. Importantly, with respect to our observations, numerous studies showed high concentrations of Wallemia spp. in outdoor and indoor air as well as in agricultural environments [33,34]. W. sebi has been frequently reported as the cause of respiratory allergies and atopic diseases in asthmatic individuals, and has also been identified as one of the causal agents in the farmer’s lung disease [35]. The second and third most prevalent fungal species, i.e., Aspergillus penicillioides and Epicoccum nigrum, have been found among the causal agents of allergic rhinitis in humans [36,37]. Out of the ten most abundant fungal genera observed in the Kraków stable, five are listed as the most common causal agents of allergies [37]. These genera are Aspergillus, Epicoccum, Trichoderma, Alternaria, and Cladosporium. Airborne fungi are described as being among the main groups of organisms involved in the pathogenesis of equine RAO [12]. Members of Aspergillus species have been detected in high concentrations in tracheal aspirates of RAO-affected animals [12]. The findings of our study corroborate this observation, as the Kraków stable has been dealing with a number of RAO cases among horses. Other fungal genera, demonstrated by Xavier et al. [12] as predominant in the respiratory tract of RAO-affected horses, included Alternaria, Cladosporium, and Penicillium. The former two (Alternaria and Cladosporium) have also been identified in our study as among the ten most abundant in the Kraków filter sample. Niedźwiedź et al. [38] examined the concentrations of allergen-specific IgE in the serum of horses suffering from RAO, and found Epicoccum nigrum (the third most prevalent species in our study) to be among the fungal species potentially involved in the occurrence of this disease.

5. Conclusions

To our best knowledge, this study is the first one to contribute, by the combination of culture-based, physicochemical, and molecular methods, to the understanding of factors affecting the occurrence of recurrent airway obstruction (RAO) in horses kept within stables. Moreover, we examined the airborne-related factors that may contribute to the occurrence of human allergic reactions and respiratory diseases, as horse stables are not only the working environment, but are also used in recreation by a variety of people. Our study demonstrated that the daily activities performed in horse stables, such as feeding or cleaning of horses, contributed largely to the increased levels of particulate matter and the fungal components of bioaerosol. The respirable fractions of fungal aerosol in many cases oscillated around 80% of the total fraction, which suggests the potential health hazard to the exposed people and animals. Out of the two examined stables in southern Poland (Kraków and Tarnów), the Tarnów stable was characterized by higher concentrations of particulate matter, whereas the total and respirable fractions of fungal aerosol were higher in the Kraków stable. Next generation sequencing conducted for the fungal community collected at the Kraków stable showed that Wallemia sebi, an important allergenic species, producing large numbers of tiny (1.5–3.0 µm) easily dispersed spores that can penetrate deeply into the respiratory tract, predominated among the identified airborne fungi. Additionally, the second and third most prevalent species, i.e., Aspergillus penicillioides and Epicoccum nigrum, are often described as causal agents of allergic reactions and potential contributors to the occurrence of respiratory diseases in horses. Having this in mind, it is highly recommended that the air quality measurements, including the assessment of particulate matter and fungal aerosol concentrations, coupled with the determination of the most prevalent fungal species, should be conducted in horse stables where there are cases of allergic reactions or respiratory diseases in workers or RAO cases among horses.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/app12189375/s1, Table S1: Relative abundance of fungal species at OTU level.

Author Contributions

Conceptualization, M.T., A.L.-B., K.K., J.K. and A.B.; methodology, A.L.-B., A.B. and K.K.; software, A.L.-B.; validation, M.T. and J.K.; formal analysis, A.L.-B.; investigation, A.B. and K.K.; resources, M.T., J.K. and A.L.-B.; data curation, K.K.; writing—original draft preparation, A.L.-B.; writing—review and editing, M.T., K.K. and J.K.; visualization, A.B.; supervision, M.T.; project administration, M.T. and J.K.; funding acquisition, M.T. and A.L.-B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the statutory measures of the University of Agriculture in Krakow and granted for the Department of Microbiology and Biomonitoring.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are entirely available in this study.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 12 09375 g001 550
Figure 1. (A). Location and schematic presentation of the Kraków stable. (B). Location and schematic presentation of the Tarnów stable.
Figure 1. (A). Location and schematic presentation of the Kraków stable. (B). Location and schematic presentation of the Tarnów stable.
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Figure 2. Share (%) of different aerodynamic diameters of airborne fungi in the Kraków (A) and Tarnów (B) horse stables at different times of the day (M—early morning-no activity; F—feeding; I.B.—indoor background, mid-day; A—afternoon, animals return; O.B.—outdoor background).
Figure 2. Share (%) of different aerodynamic diameters of airborne fungi in the Kraków (A) and Tarnów (B) horse stables at different times of the day (M—early morning-no activity; F—feeding; I.B.—indoor background, mid-day; A—afternoon, animals return; O.B.—outdoor background).
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Figure 3. Relative abundance (%) of fungal species, the share of which in the total number of reads exceeded 0.2% (A) and 10 most abundant fungal genera in the examined sample (B).
Figure 3. Relative abundance (%) of fungal species, the share of which in the total number of reads exceeded 0.2% (A) and 10 most abundant fungal genera in the examined sample (B).
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Figure 4. Concentration of total fungal aerosol (CFU/m3) in different times of day compared with the inhalable particulate matter concentration (PM10; µg/m3). Kraków (A) and Tarnów (B) horse stables.
Figure 4. Concentration of total fungal aerosol (CFU/m3) in different times of day compared with the inhalable particulate matter concentration (PM10; µg/m3). Kraków (A) and Tarnów (B) horse stables.
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Figure 5. Concentration of respirable fungal aerosol (CFU/m3) at different times of the day compared with the concentration of respirable particulate matter (µg/m3). Kraków (A) and Tarnów (B) horse stables.
Figure 5. Concentration of respirable fungal aerosol (CFU/m3) at different times of the day compared with the concentration of respirable particulate matter (µg/m3). Kraków (A) and Tarnów (B) horse stables.
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Table
Table 1. Mean values of particulate matter concentration (µg/m3) along with temperature (°C) and relative humidity (%) in the examined horse stables and outdoors. The presented values are means of six replicates from one hour intervals. Values in brackets show standard deviation. The highest values recorded indoors are shown in bold.
Table 1. Mean values of particulate matter concentration (µg/m3) along with temperature (°C) and relative humidity (%) in the examined horse stables and outdoors. The presented values are means of six replicates from one hour intervals. Values in brackets show standard deviation. The highest values recorded indoors are shown in bold.
Time of DayEarly Morning
(No Activity)		FeedingMid-DayActivityOutdoor Background
Horse StablesKrakówTarnówKrakówTarnówKrakówTarnówKrakówTarnówKrakówTarnów
PM1019 (1)54 (1)16 (1)57 (1)10 (1)62 (1)19 (1)109 (2)28 (2)36 (0)
PM2,513 (1)46 (1)14 (1)47 (0)9 (1)51 (1)18 (1)86 (5)20 (1)32 (1)
PM16 (1)29 (1)6 (1)29 (1)5 (0)32 (2)10 (1)33 (3)15 (2)20 (0)
Respirable dust19 (2)74 (0)19 (1)76 (1)14 (1)82 (2)27 (1)119 (8)35 (1)52 (1)
Temperature73871188752
Relative humidity70606371555364626972
Table
Table 2. Concentration of fungal aerosol (CFU/m3) in the examined horse stables and outdoors. The presented values are means of three replicates. Values in brackets show standard deviation. The highest values within each fraction of bioaerosol are shown in bold.
Table 2. Concentration of fungal aerosol (CFU/m3) in the examined horse stables and outdoors. The presented values are means of three replicates. Values in brackets show standard deviation. The highest values within each fraction of bioaerosol are shown in bold.
Time of DayEarly Morning
(No Activity)		FeedingMid-DayActivityOutdoor Background
Horse StablesKrakówTarnówKrakówTarnówKrakówTarnówKrakówTarnówKrakówTarnów
Fraction F1 (7.0–11.0 µm)541 (1)701 (300)2.82 × 104 (34)336 (336)422 (2)1.65 × 103 (654)901 (1)3.53 × 103 (748)0 (0)128 (128)
Fraction F2 (4.7–7.0 µm)212 (12)654 (395)2.41 × 104 (617)830 (18)70 (0)860 (53)858 (8)3.59 × 103 (306)12 (2)44 (0)
Fraction F3 (3.3–4.7 µm)436 (36)925 (571)1.90 × 104 (41)601 (247)173 (3)931 (365)1.33 × 103 (30)3.82 × 103 (707)12 (4)22 (22)
Fraction F4 (2.1–3.3 µm)594 (6)306 (118)1.57 × 104 (201)2.31 × 103 (371)281 (1)2733 (59)1.94 × 103 (39)3.98 × 103 (353)59 (4)146 (0)
Fraction F5 (1.1–2.1 µm)5.65×103 (148)347 (206)1.67 × 104 (231)3.32 × 103 (671)373 (3)2.73 × 103 (283)6.71 × 103 (7)3.60 × 103 (1060)177 (3)146 (66)
Fraction F6 (0.65–1.1 µm)33 (3)141 (47)8.24 × 103 (240)353 (0)46 (1)471 (165)582 (2)4.57 × 103 (1458)0 (1)13 (13)
Total fungal aerosol7.46 × 103 (193)3.07 × 103 (989)11.21 × 104 (1364)7.76 × 103 (1148)1.37 × 103 (12)8.44 × 103 (1926)1.23 × 104 (86)2.31 × 104 (2058)259 (9)406 (230)
Respirable fungal aerosol6.71 × 103 (180)1.72 × 103 (294)5.97 × 104 (712)6.59 × 103 (795)874 (9)5.93 × 103 (872)10.56 × 103 (78)15.96 × 103 (1458)247 (10)234 (102)
Table
Table 3. Spearman’s correlation coefficient matrix for microbial bioaerosol components, PM fractions, temperature, and relative humidity of air. Bolded values are significant at p < 0.05.
Table 3. Spearman’s correlation coefficient matrix for microbial bioaerosol components, PM fractions, temperature, and relative humidity of air. Bolded values are significant at p < 0.05.
Kraków Stable
Fungal AerosolParticulate Matter (PM)Microclimatic
Parameters
F1F2F3F4F5F6TotalRespirablePM1PM2.5PM10RespirableTemp.Relative Humidity
F1-0.9971.0000.9850.9970.9020.9970.997−0.297−0.153−0.319−0.1530.347−0.203
F2 -0.9970.9761.0000.9000.9881.000−0.296−0.152−0.318−0.1520.346−0.202
F3 -0.9850.9970.9020.9970.997−0.297−0.153−0.319−0.1530.347−0.203
F4 -0.9760.8880.9880.976−0.334−0.183−0.343−0.1830.346−0.202
F5 -0.9000.9881.000−0.296−0.152−0.318−0.1520.346−0.202
F6 -0.9000.900−0.372−0.287−0.564−0.2870.650−0.597
Total fungal aerosol -0.988−0.296−0.152−0.318−0.1520.346−0.202
Respirable fungal aerosol -−0.296−0.152−0.318−0.1520.346−0.202
PM1 -0.9690.8700.969−0.6410.433
PM2.5 -0.9021.000−0.7220.500
PM10 -0.902−0.8960.786
Respirable PM -−0.7220.500
Temperature -−0.860
Relative humidity -
Tarnów Stable
Fungal AerosolParticulate Matter (PM)Microclimatic
Parameters
F1F2F3F4F5F6TotalRespirablePM1PM2.5PM10RespirableTemp.Relative Humidity
F1-0.8630.8390.6190.4560.7590.7780.7420.7940.6740.6440.6770.716−0.827
F2 -0.9360.8050.6870.9090.9120.8880.7510.7130.6750.6950.849−0.765
F3 -0.7290.5030.8570.8420.8060.6500.6630.7030.6200.808−0.812
F4 -0.8450.9450.9480.9730.6950.6770.6440.6890.963−0.593
F5 -0.8210.8300.8420.7240.6990.5930.7230.821−0.345
F6 -0.9970.9790.7940.7930.7850.7870.963−0.642
Total fungal aerosol -0.9880.8040.7900.7710.7900.960−0.640
Respirable fungal aerosol -0.7490.7360.7030.7420.960−0.615
PM1 -0.9110.8390.9350.799−0.673
PM2.5 -0.9630.9940.811−0.494
PM10 -0.9420.777−0.484
Respirable PM -0.811−0.494
Temperature -−0.667
Relative humidity -
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Lenart-Boroń, A.; Bajor, A.; Tischner, M.; Kulik, K.; Kabacińska, J. Particulate Matter Concentrations and Fungal Aerosol in Horse Stables as Potential Causal Agents in Recurrent Airway Disease in Horses and Human Asthma and Allergies. Appl. Sci. 2022, 12, 9375. https://0-doi-org.brum.beds.ac.uk/10.3390/app12189375

AMA Style

Lenart-Boroń A, Bajor A, Tischner M, Kulik K, Kabacińska J. Particulate Matter Concentrations and Fungal Aerosol in Horse Stables as Potential Causal Agents in Recurrent Airway Disease in Horses and Human Asthma and Allergies. Applied Sciences. 2022; 12(18):9375. https://0-doi-org.brum.beds.ac.uk/10.3390/app12189375

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Lenart-Boroń, Anna, Anna Bajor, Marek Tischner, Klaudia Kulik, and Julia Kabacińska. 2022. "Particulate Matter Concentrations and Fungal Aerosol in Horse Stables as Potential Causal Agents in Recurrent Airway Disease in Horses and Human Asthma and Allergies" Applied Sciences 12, no. 18: 9375. https://0-doi-org.brum.beds.ac.uk/10.3390/app12189375

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