Mycological air contamination level and biodiversity of airborne fungi isolated from the zoological garden air — preliminary research

Abstract   The aim of this paper was to evaluate the degree of mycological air contamination and determine the taxonomic diversity of airborne fungi residing in the air of 20 different animal facilities in a zoological garden. The concentrations of fungi in the zoological garden were measured using a MAS-100 air sampler. The collected microorganisms were identified using the combination of molecular and morphological methods. The fungal concentration ranged from 50 to 3.65 × 104 CFU/m3 during the whole study. The quantitative analysis of the fungal aerosol showed that the obtained concentration values were lower than the recommended permissible limits (5 × 104 CFU/m3 for fungi). Environmental factors, including temperature and relative humidity, exerted a varying effect on the presence and concentration of isolated fungi. Relative humidity was shown to correlate positively with the concentration of fungal spores in the air of the facilities studied (rho = 0.57, p < 0.0021). In parallel, no significant correlation was established between temperature and total fungal concentration (rho =  − 0.1, p < 0.2263). A total of 112 fungal strains belonging to 50 species and 10 genera were isolated. Penicillium was the dominant genera, including 58.9% of total fungal strains, followed by Aspergillus 25.89%, Cladosporium 3.57%, Talaromyces 3.57%, Mucor 1.78%, Schizophyllum 1.78%, Syncephalastrum 0.89%, Alternaria 0.89%, Absidia 0.89%, and Cunninghamella 0.89%. Our preliminary studies provide basic information about the fungal concentrations, as well as their biodiversity in zoological garden. Further studies are needed to generate additional data from long-term sampling in order to increase our understanding of airborne fungal composition in the zoological garden. Supplementary Information The online version contains supplementary material available at 10.1007/s11356-024-33926-2.


Introduction
Zoological gardens are one of the most popular attractions visited by tourists worldwide.We should perceive them not only as places where a large diversity of animals is kept, but also as places where people can admire both native and exotic species (Nekolný and Fialová 2018).Both visitors and zoo workers are potentially exposed to bioaerosols which contain bacteria, viruses, pollens, fungi, and mycotoxins (Michalska et al. 2021;Nageen et al. 2023).Among the microorganisms in bioaerosols, fungi are the most numerous group of biological particles (Szulc et al. 2020).Those microorganisms are abundant in the environment and play important roles as symbionts, saprotrophs, or parasites.It is estimated that airborne fungi constitute nearly 25% of the global biomass and, therefore, play a significant role in air pollution affecting human health (almost 150 fungal taxa are associated with allergies) (Nageen et al. 2023).
National and global research about the contamination and the biological diversity of fungi in breeding facilities was carried out mainly in the large-scale poultry houses, barns, and piggeries (Plewa and Lonc 2011;Pusz et al. 2015;Douglas et al. 2018;Seifi et al. 2018;Lee and Kim 2021).Against this background, research on fungal biodiversity and the degree of air and environmental pollution in zoological gardens are scarce (Rivas et al. 2018;Cateau et al. 2022;Álvarez-Pérez et al. 2023;Debergh et al. 2023).
The majority of these cited works primarily concentrate on fungi of the genus Aspergillus, with a particular focus on Aspergillus fumigatus, its sensitivity to azole drugs, and the detection of fungi solely in the environment of a single animal group (penguins).They do not usually consider the presence of fungi in other animal habitats and another group of molds.Moreover, the only national study on microbial air contamination was carried out in Krakow's Zoo (Grzyb andLenart-Boroń 2019, 2020).The research focused entirely on the determining occurrence of individual bioaerosol (bacterial and fungal) fractions, without taking into account the biodiversity of fungi and their potential toxicity.The studies conducted in animal breeding places other than zoos often showed high concentrations of this group of microorganisms that can be harmful to human and animal health.Among the isolated fungi, the most frequently identified were fungi belonging to the genera Aspergillus (A. fumigatus, A. niger, A. flavus), Penicillium (P.citrinum, P. viridicatum), Cladosporium spp., Alternaria spp., and Scopulariopsis spp.(Plewa and Lonc 2011;Pusz et al. 2015;Seifi et al. 2018).The presence of these poses a risk of disease in people with bronchial asthma, EAA (extrinsic allergic alveolitis), allergic rhinitis, and ODTS (dust-induced toxic syndrome organic), while in animals, it causes pulmonary aspergillosis and mycotoxicosis (Szulc et al. 2020).
Therefore, research in zoological gardens is important because these facilities are not only places for breeding animals but may be a source of dangerous fungi.This is important information, taking into account the specificity of zoos, which are not only a working environment, but also one of the most frequently visited tourist places.Thus, the aim of this paper was to evaluate the degree of mycological air contamination and determine the taxonomic diversity of airborne fungi residing in the air of different animal facilities using the combination of microscopic and genetic analyses.

Study area
The study was conducted at the Zoological Garden in Wroclaw.This zoo has the largest collection of animals in Poland, with almost 1100 different species in an area covering 33 hectares (https:// zoo.wrocl aw.pl).
The measurements were carried out inside twenty facilities: two sites of Monkey House, four sites of Apes Pavilion (Pan troglodytes), Papio Pavilion (Papio anubis), three cages with maggots (Macaca sylvanus), five sites in Kongo Pavilion (with crocodiles, manatees, and numerous species of birds), and five sites in East Africa (with the Hippopotamus amphibious, Orycteropus afer, Heterocephalus glaber).
The choice of study sites was guided by their convenient accessibility.

Sampling methods
Air samples were taken in October 2022 and in January, April, and June 2023 using a MAS-100 air sampler (Merck KgaA, Darmstadt, Germany).Three parallel samples (two incubated at 27 °C degree, one at 37 °C) were collected at the center point of each location at a height of 1.5 m above the ground and directly struck on the surface of Sabouraud agar.The plates were incubated for about 7 days at 25 °C (two samples) and 37 °C (one sample).The number of fungal colonies was expressed as a total colony-forming units (CFU/m 3 ) and revised by the equation: where Pr, r, and N stand for revised colonies, number of viable colonies, and the number of sieve pores, respectively (Feller 1968).The concentration of airborne microorganisms (CFU/m 3 ) was calculated according to the following formula: X = (a × 1000)/V, where "a" is the number of fungal colonies, and "V" is the air volume sampled (m 3 ) (Michalska et al. 2021).

Characterization of meteorological conditions
The air temperature and relative humidity were measured during each sampling session using a thermo-hygrometer (HI9565 HANNA, Poland).The air temperature ranged from 11.2 to 22.7 °C (autumn season), 17.1 to 24 °C (winter season), 18.1 to 24.6 °C (spring season), and 19.4 to 25.6 °C (summer season), respectively.Relative humidity in the autumn season was between 27.9 and 89.4%, in the winter season from 30.8 to 95.5%, in the spring season from 35.5 to 90.8, and in the summer season from 54.7 to 80.1%.

Identification of fungi
Airborne fungi were identified based on their macro-and microscopic features using diagnostic keys (Samson et al. 2014(Samson et al. , 2019;;Yilmaz et al. 2014;Visagie et al. 2014).Then, the cultured fungi were subjected to molecular identification to confirm species identity.DNA extraction from the selected and dominant fungi was performed using the Tissue DNA Purification Kit (EURx, Gdańsk, Poland) according to the manufacturer's instructions.For the diagnostic of the airborne fungi, we used both morphological criteria and molecular analyses based mainly on the sequence of the internal transcribed spacer ITS.In the case of several closely related strains, we sequenced b-tubulin and calmodulin fragment.Amplification parts of the ITS were amplified according to the methods described by White et al. (1990).The ITS was amplified using a pair of primers: ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′).PCR reactions were performed in a T100 Thermal Cycler (Bio-Rad, Warsaw, Poland) in a total volume of 12.5 µL.Each PCR reaction contains 6.25 μL of 2 × PCR Mix Plus (A&A Biotechnology, Gdansk, Poland), 0.625 μL of each primer (10 mM), 4 μL of DNA template, and 1 μL of ddH 2 O. PCR conditions included an initial denaturation step of 95 °C (30 s); 34 cycles of 95 °C (45 s), 55 °C (60 s), and 72 °C (60 s); and the final elongation of 72 °C (3 min).
All positive samples obtained in the PCR reaction were purified and sequenced (Macrogen, Amsterdam, the Netherlands) using the same primer pairs as in the PCR reaction.The obtained sequences were manually edited using DNA Baser Sequence Assembly software (Heracle BioSoft SRL Romania), and consensus sequences were aligned and compared with sequences deposited in the National Center for Biotechnology Information's GenBank (NCBI, Bethesda, MD, USA) using the BLAST algorithm (http:// www.ncbi.nlm.nih.gov/).The most representative sequences that form the basis of the phylogenetic tree have been included in the GenBank database (Table 1).Phylogenetic analysis was performed using the Maximum Likelihood method with the MEGA 7.0 software, bootstrapping was performed using 1000 replicates.

Statistical analysis
The data underwent statistical analysis using R version 4.3.1 in RStudia 2023.09.0.Linear regression models and Spearman correlations were computed to evaluate the correlation between fungal abundance and meteorological parameters (humidity and temperature).The Kruskal-Wallis test and Dunn's post hoc test for multiple comparisons were utilized to investigate the influence of qualitative factors, such as location and season, on fungal abundance.For statistical significance, results with p < 0.05 were considered.Margalef's Index and Jaccard's Similarity Index were employed to assess the diversity of fungal species at specific research sites.The Jaccard Index was calculated pairwise, and then the average value for each site was obtained.

Fungal concentrations in air samples
A total of 240 air samples were collected from 20 locations in the zoological garden.Fungi were detected in 234 (97.5%) samples.The concentrations of countable fungal aerosol are presented in Table 2 and ranged from 5.0 × 10 1 to 3.65 × 10 4 CFU/m 3 (for a temperature of 27 °C).The lowest concentration of fungi was recorded in Papio Pavilion 5 × 10 1 CFU/m 3 (range 5 × 10 1 -2.5 × 10 2 CFU/m 3 ).It is worth noticing that this concentration was lower than the recorded at another site, because the air sampling was done just before the morning cleaning of the room.The highest concentration of fungi was recorded in the Apes Pavilion and ranged between 2.02 × 10 4 and 3.65 × 10 4 CFU/m 3 .Similar concentrations were found in the Papio Pavilion (range 1.31 × 10 4 -1.32 × 10 2 CFU/m 3 ) and the Kongo Pavilion (1.38 × 10 4 -2.61 × 10 4 CFU/m 3 ).Statistical analysis performed using the Kruskal-Walis test showed statistically significant differences between the sampling location and the CFU/m 3 fungi values (Fig. 1).
There have been a lot of studies about microbial and mycological air contamination in animal production premises (Plewa and Lonc 2011;Pusz et al. 2015;Matković  .The air quality in some farming objects has been described by Radon et al. (2002).According to that author, the total number of fungi in poultry houses and in pig farms was higher than the concentrations of airborne fungi noticed in our research and ranged from 8.3 × 10 4 to 1.1 × 10 9 (Radon et al. 2002).Such large differences in the level of microbial contamination between the zoological garden and other breeding premises are most likely due to different methods of air sampling.The sampler used in our investigation has some limitations.This device is characterized by sampling efficiency described by the cuttoff size parameter D50, at the level of 1.7 μm (Górny et al. 2023).All particles above the size would be collected and almost all particles smaller than that size may not be captured by this impactor.Airborne fungal spores typically have an aerodynamic diameter (d ae ) of 2 to 4 μm, and some species release fragments of 0.3 to 1.3 μm (Madsen et al. 2016).That is why the number of airborne fungi in our research may have been underestimated in relation to the actual concentration in the air.The results of the present study suggest that it is necessary to use the same instruments and methods to assess microbial contamination.
The lack of standardization of air sampling methods may lead to incorrect conclusions.The concentration of airborne fungi varies across different seasons and is dependent on environmental factors such as temperature or relative humidity (Yuan et al. 2022).The important factors that increase the spread of fungi in the air and sustain their growth are temperature and humidity (Pflieger et al. 2020).Therefore, our studies were conducted in a seasonal cycle (spring, summer, autumn, winter), and the environmental conditions (temperature and relative air humidity) were measured during each sampling season using a thermo-hygrometer (Table 3).The lowest recorded temperature (11 °C) was noted in autumn in the room for Papio Pavilion, while the highest temperatures were noted in summer in the Kongo Pavilion (25.5-25.9°C).Similarly, the highest humidity was observed in winter in East Africa (95.5%) and in the Kongo Pavilion.In general, the relative humidity during the studies varied from 29.3% (in the Apes Pavilion) to 95.5% in the Kongo Pavilion.Environmental parameters (temperature and relative humidity) were correlated with the total number of CFU/m 3 noted in all locations, based on Spearman correlation analysis.The relative humidity correlated positively with the total fungal concentration (rho = 0.57, p < 0.0021), while temperature showed no significant correlation with the total fungal concentration at all locations (rho = − 0.1, p < 0.2263) (Figs. 2 and 3).The concentration of airborne fungi observed at each location varied significantly across the seasons.The highest concentration was recorded in the autumn, reaching a level of 3.65 × 10 5 CFU/m 3 while the lowest levels were observed in the spring and summer, with values of 1.5 × 10 4 and 1.32 × 10 4 CFU/m 3 , respectively.The lowest concentration of fungi (2.5 × 10 2 CFU/m 3 ) was observed in the winter season.It has been demonstrated that optimal temperature and high relative humidity can contribute to a sudden increase in the concentration of airborne fungi.The medium fungal concentrations varied considerably by season, with the greatest variation noted between summer and autumn (p < 0.0018).A strong variety of airborne fungal concentrations was also noticed between spring and summer (p < 0.0056), while no statistically significant differences were observed between other seasons (Fig. 4).
Interpretation of the results of microbiological air contamination is challenging because of the lack of acceptable Fig. 2 The correlation between temperature and fungal conidia concentration in the air Fig. 3 The correlation between relative humidity and fungal conidia concentration in the air limits for microbiological agents.The most commonly used measure of exposure to microbial air pollution is the degree of such pollution, expressed in terms of the number of colony-forming units (CFU) in 1 m 3 of air (Górny et al. 2016).The threshold limit values (TLV) were used in the assessment of mycological air contamination.For microbiological agents in the air of occupational and non-occupational environments, TLV was proposed by the Expert Group on Biological Agents at the Polish Interdepartmental Commission for Maximum Admissible Concentrations and Intensities for Agents Harmful to Health in the Working Environment (Górny et al. 2016).These values (5 × 10 4 CFU/m 3 for fungi) were developed as a result of volumetric measurements of environmental bioaerosols.Based on the TLV proposed by the mentioned Expert Group, we can conclude that the quantitative analysis of the fungal aerosol showed lower concentration values than the recommended permissible limits.Nevertheless, this is not equivalent to the absence of microbial contamination in the facilities that were studied.Particularly as some of the values were close to TLV values.The US government agency, Occupational Safety and Health Administration (OSHA), suggests that a value higher than 1.0 × 10 3 CFU/m 3 indoor may be an indicator of microbial contamination.However, a determination of bioaerosol biodiversity is needed to confirm a health hazard, as certain species may pose a greater health concern than others (OSHA 2015;Rivas et al. 2018).

Biodiversity of airborne fungi
The total number of airborne fungi varied by location (Table 4) and seasons (Table 5).The Margalef's Index was used to analyze the biodiversity of airborne fungi throughout the study period and in different locations.The highest value of Margalef's Index (0.93) was recorded at Kongo Pavilion in location no 18 and the lowest (0.00) was observed at East Africa Pavilion in location no 12.Concerning seasons, the highest diversity of airborne fungi was recorded in the winter (34), followed by the autumn (29), and spring ( 27), whereas the lowest number of strains were noted in the summer ( 21).The average Jaccard Index for all sites was quite low, at 0.135.The lowest average Jaccard Index, calculated pairwise for each research site, was for the Kongo Pavilion (0.12).This indicates that the Kongo Pavilion had the highest number of unique isolated species compared to other research sites.Direct comparison showed the lowest species similarity between the Kongo Pavilion and the Apes Pavilion (Jaccard Index = 0.06) and between the Kongo Pavilion and the Papio Pavilion (0.08).The highest similarity was found between the Kongo Pavilion and the East Africa Pavilion (0.22).
We used both morphological and molecular identity methods based on sequencing regions of the ITS, b-tubulin, and calmodulin.Using these methods, a total of 112 fungal strains belonging to 10 genera and 50 species were isolated during the whole 1-year-long study.The molecular identification corresponded well with the morphological diagnosis and proved to be a good tool for diagnostic strains that were not identified microscopically.By comparing the partial sequences of the ITS region of the own isolates with the sequences of other isolates available in GenBank, the results showed that the similarity percentage of nucleotide sequences ranged between 95 and 100% (Supplement 1).In most cases, (87%) the sequence data allowed the identification at the species level.The study based on the molecular analysis allowed us to identify 97 out of 112 species of fungi.Diagnosis of thirteen strains belonged to Penicillium genera, and one strain each of the Cladosporium and Absidia genera failed.Analysis of ITS region revealed that they belong to closely related species.Even the sequencing of the less conservative region (b-tubulin) and calmodulin did not allow for the identification of these fungi at the species level.
Diversity of identified fungi varied between different locations and seasons.Unfortunately, there is a lack of research about the biodiversity of airborne fungi in zoological gardens.Therefore, our results could be compared to the studies obtained for the livestock premises.For instance, in the China poultry houses, researchers observed a different genus composition than in our studies.They isolated mainly Trichosporon, Candida, Aspergillus, Cladosporium, and Alternaria genera (Chen et al. 2021).In other studies, Aspergillus, Scopulariopsis, Penicillium, and Cladosporium were detected in the air of the swine house (Kumari et al. 2016).Comparable results to ours have been reported by Matković et al. 2009.Croatian authors detected Aspergillus and Penicillium genera in dwellings for dairy cows and laying hens.
Some of the fungi isolated in our research may be associated with allergic respiratory diseases (especially in people with a weakened immune system).Filamentous fungi can be a source of polysaccharides such as the β(1 → 3)-glucans.This compound may cause inflammatory airway reactions and also affect the immune system.There is increasing evidence that β(1 → 3)-glucans cause non-specific inflammatory reactions and can be responsible for bioaerosol-induced respiratory symptoms observed in both indoor and occupational environments (Pflieger et al. 2020).The other fungi are important producers of mycotoxins, which have been suggested as one of the major possible causes of health problems.For example, several detected fungi belonging to   Aspergilli in sections Circumdati (A. steynii and A. westerdijkiae), Flavi (A. flavus), and Nigri (A. carbonarius and A. niger) are well-known producers of ochratoxins, a mycotoxin teratogenic, carcinogenic, immunosuppressive, and nephrotoxic in animals (Kagot et al. 2019).The other species, such as A. versicolor and A. flavus, produce sterigmatocystin and aflatoxins, respectively.Aflatoxins are the most potent natural carcinogens (Lamoth 2016).What's more, the identified in our study A. flavus is recognized as an opportunistic animal and human pathogen, while A. fumigatus is the main causative agent of pulmonary invasive aspergillosis (Al-Shaarani et al. 2023).The most abundant Penicillium genera isolated in our studies is listed among the most common allergenic fungal taxa and has been linked to asthma.Whereas, Schizophyllum commune, which accounted for 1.78%, may be the cause of ABPM (allergic bronchopulmonary mycosis) and allergy-related bronchopulmonary infections and sinusitis (Oguma et al. 2023).

Conclusion
This is the first research on culturable airborne fungi at the zoological garden in Poland.The quantitative analysis of the fungal aerosol showed that the obtained concentration values were lower than the recommended permissible limits.But a lot of detected fungi can be harmful to human and animal health.That is why, our study emphasizes the necessity of air quality monitoring.Similar studies to ours are rarely conducted.Therefore, our preliminary research provides basic information about the fungal concentrations and their biodiversity in these touristic facilities.However, further long-term quantitative, as well as qualitative, and mycotoxicological research is needed to fully understand airborne fungal composition in the zoological garden and its potentially negative impact on human and animal health.Moreover, quantitative and qualitative assessment of mycotoxins in the air is of great importance from the occupational exposure point of view.In addition, our preliminary results along with planned long-term studies of mycological air contamination in the zoo may contribute in the future to the development of microbiological air quality standards by relevant institutions.

Fig. 6
Fig. 6 Maximum likelihood (ML) phylogenetic tree (Jukes-Cantor + G) for Aspergillus sp.based on sequences of the ITS gene fragment.Bootstrap values are shown above the branches.Sequences from this study are marked with solid circles.The dendrogram was constructed with 1000 replications using MEGA software

Table 1
Molecular identification (using ITS region) of fungi isolated from the air in the zoological garden

Table 2
Matković et al. (2007) other studies on the mycological quality of air in zoos, the results of our own research can be compared to other breeding facilities, e.g., poultry houses and cowshed.Matković et al. (2007)observed in the barn the average concentration of fungi ranged from 5.23 × 10 4 CFU/m 3 (at noon) to 8.35 × 10 4 CFU/m 3 (in the morning)

Table 5
Number of fungi species isolated from the air in the seasons