Effects of Season and House Microclimate on Fungal Flora in Air and Broiler Trachea

: Fungi are present in abundance in poultry housing. The aim of the study was to assess the effect of season and microclimate parameters in poultry housing on fungal ﬂora in the air and broiler trachea in commercial fattening conditions. The study was conducted in summer and winter. Study results indicated seasonal impact and association between fungal ﬂora composition in housing air and broiler trachea. However, the total fungal count in housing air was signiﬁcantly higher in summer and in broiler trachea in winter, both signiﬁcantly correlated with indoor relative humidity and ammonia concentration. There was no signiﬁcant correlation between outdoor and indoor air temperature, relative humidity and airﬂow rate, respectively. Study results suggested that environmental determination of fungi should be accompanied by their determination in broilers. In addition, seasonal impact on fungal contamination should be associated with microclimate conditions in the poultry house rather than the season itself. The fungi detected and the results obtained have implications not only for broiler health but also for the health of humans working in such environments.


Introduction
Diseases occurring in intensive livestock production that are directly associated with the environment of animal farming are influenced by a number of factors including air quality. Fungal spores are a constituent of bioaerosol. Comparative studies of air quality in housing intended for various farm animals have shown that poultry housing air contains the highest rate of fungi [1,2], broiler houses in particular [3,4]. Like bacteria, fungi may originate from the soil, dust, feed and litter, and to a lesser extent from the poultry [5], although some species such as Aspergillus sp. may utilise and degrade keratin from feathers [6]. Although fungal diseases are less common in poultry as compared with bacterial and viral diseases, they should not be neglected because when occurring, they can cause considerable economic losses either by direct infestation or via mycotoxin production [7].
Moulds of the genus Aspergillus are ubiquitous saprophytic microorganisms that induce clinically manifest infections in poultry under specific conditions, primarily involving respiratory system [8]. Aspergillosis is a severe disease in poultry farming all over the world [9], associated with high morbidity and mortality, along with poor feed conversion and weight gain in recovering birds, and carcass condemnation [7,10]. Young birds are more susceptible to acute aspergillosis, whereas chronic form of the disease is sporadic and more common in older poultry. Although the chronic form is by far less frequent, it causes enormous losses by affecting older and thus economically higher-price poultry [10,11]. The disease is also important from the occupational and public health aspects [12][13][14].
Aspergillosis is mostly caused by A. fumigatus, the most pathogenic fungus affecting poultry [7,15], however, the role of other Aspergillus species in the disease should not be ignored [9]. Aspergillosis is not a contagious disease but develops upon inspiring a large number of spores, or in cases of reduced resistance in poultry. The factors favouring disease development include long-term exposure and heavily contaminated environment, stress, immunosuppression, poor ventilation and sanitation, wet litter, malnutrition, and prolonged feed storage [8,16]. Season and microclimate parameters are known to be important factors that influence the occurrence of fungi and fungal diseases in poultry production. However, there is no strict consensus on their effects. Sajid et al. [17] report on a higher incidence of aspergillosis in poultry during warm and humid season as compared with cold season, which is consistent with the results reported by Sultana et al. [18]. Viegas et al. [19] found that fungal air contamination in poultry houses increased with higher indoor air temperature, whereas the study conducted by Popescu et al. [20] showed negative correlation of fungal count in the house air with air temperature but positive correlation with relative humidity inside houses. Lawniczek-Walczyk et al. [21] recorded no significant correlation of indoor air temperature or relative humidity with fungal count in the house air, and no seasonal differences in fungal count. Debey et al. [22] reported a higher yeast count in poultry house air in winter as compared with summer, whereas the total mould count showed no significant seasonal difference. However, seasonal differences were recorded in particular mould counts, with higher Aspergillus sp. concentration recorded in winter as compared with summer. The increased rate of Aspergillus sp. isolation correlated negatively with relative humidity. Wójcik et al. [23] found a higher fungal count in poultry house air in winter as compared with summer.
The aim of the study was to compare mycoflora in the house air and broiler trachea between summer and winter, with special reference to Aspergillus sp. The following hypotheses were tested: (i) fungal count and composition in poultry house air will differ between summer and winter, (ii) fungal flora in poultry house air will be correlated with fungal flora in broiler trachea, (iii) indoor microclimate conditions will influence fungal flora, and (iv) outdoor climate conditions will influence indoor microclimate.

Location and Climate
The study was carried out in Koprivnica-Križevci County, Republic of Croatia, characterized by temperate continental climate with precipitation spread throughout the year, 850-900 mm on average. There are two precipitation peaks, in July and November, whereas February has the lowest precipitation level. The highest summer temperatures are accompanied by the highest precipitation levels. There are no extremely dry periods. Winds blow throughout the year, thus the area is characterised by gentle winds. Maximal air humidity is recorded in November and December, and minimal humidity in April and May, with 82% mean annual relative humidity [24].

Experimental Design
The study included broiler fattening cycles in summer (July-August 2016) and winter (December-January 2016/2017). In each season, fattening cycle lasted for five weeks, with 18,000 Ross hybrid broilers kept under commercial housing conditions in a closed house with controlled microclimate, at up to 33 kg/m 2 stocking density, and a mixture of chopped straw and sawdust (10 cm depth) used as litter. Heating was provided by oil heater and ventilation by a negative pressure system. The lighting programme was set in accordance with the Ross broiler manufacturer's recommendations [25]. Broilers were fed complete feed mixture from round pan feeders and watered from nipple drinkers with cups, with ad libitum feeding and watering. The broiler house is cleaned and disinfected after each production cycle, with two-week house rest between the cycles.
Microclimate conditions, air temperature, relative humidity and airflow rate inside the house, ammonia concentration in house air, and fungal concentration in house air and in broiler trachea were determined once a week during fattening period in both seasons. Air temperature, relative humidity and airflow rate were also measured at 5 m outdoor weekly. Air temperature ( • C), relative humidity (%), airflow rate (m/s) and ammonia concentration (ppm) were measured by portable digital instruments (Testo SE & Co. KGaA, Lenzkirch, Germany, and Dräger Safety AG & Co. KGaA, Lübeck, Germany). For determination of fungal count, the air was sampled onto Petri dishes with Sabouraud dextrose agar (Biolife, Milan, Italy) using a SAS 100 TM device (PBI International, Milan, Italy). Plates were incubated at 25 • C for five to seven days and grown colonies were expressed as colony-forming units per m 3 air (CFU/m 3 ), with result correction according to the table and formula supplied with the device. Both indoor and outdoor measurements were performed from 9.00 to 12.00 a.m. at nine sites. The presence of fungi in broilers was determined in tracheal swabs obtained weekly from 30 randomly chosen, apparently healthy birds. Tracheal swabs were taken with a sterile stick soaked with sterile saline (1 mL), then 100 µL was smeared on agar. Agar and incubation conditions were the same as for airborne fungi. Results were expressed as CFU/swab. Fungal identification was carried out by macroscopic observation of grown colonies and microscopic examination of spores using lactophenol blue stain [26].

Statistical Analysis
Data analysis was performed by Statistica v. 13.5 reference software (TIBCO Software Inc., Palo Alto, CA, USA, 2018). Data were processed by standard descriptive statistics methods and expressed as mean and standard deviation or median and minimum-maximum values, depending on the normality of distribution estimated by the Kolmogorov-Smirnov test. Student's t-test was used to test the significance of differences in the mean values of air temperature, relative humidity and airflow rate inside and outside the house, ammonia concentration in house air, and total fungal count in house air between two seasons. The significance of differences in the median count of total fungi in broiler trachea and particular fungal counts in house air and broiler trachea between the seasons were tested by Mann-Whitney U-test, and so were between-season differences in the values of all parameters observed according to fattening weeks. Differences among fattening weeks within a season were tested by Friedman ANOVA and Wilcoxon matched pairs test for post hoc analysis. Correlations among study parameters were assessed by Spearman rank order correlations. The level of statistical significance was set at p < 0.05, although differences at the levels of p < 0.01 and p < 0.001 are also reported in tables.

Results and Discussion
Study results on the average values of air temperature, relative humidity and airflow rate inside and outside the house, ammonia concentration in house air, and fungal count in house air and broiler trachea (total and particular fungal count) in summer and winter, total fungal count in house air and broiler trachea according to fattening weeks in each season, and correlations among study parameters are shown in Tables 1-6 and Figures 1 and 2. The figures showing indoor and outdoor air temperature, relative humidity and airflow rate, and ammonia concentration in house air according to fattening weeks in each season are presented in Supplementary Materials. During the study period, no cases of fungal diseases were recorded, while the rate of broiler deaths was within the technologically predicted rate in both seasons.  Values in the same row differ significantly at ** p < 0.01. Table 4. Correlation between house microclimate parameters, and airborne and tracheal fungi.  Table 5. Composition of airborne fungal flora in broiler house during five-week fattening in summer and winter.

Figure 2.
Tracheal fungi according to broiler fattening weeks in summer and winter. a,b,c All values within the same season differ significantly (p < 0.05), except for those marked with the same letter. All values within the same weeks differ significantly between the seasons (p < 0.05).
Mean air temperature outside the house was significantly higher (p < 0.05) during fattening period in summer, whereas the mean relative humidity and airflow rate were significantly higher (p < 0.05) in winter ( Table 2). There was no significant correlation between either outdoor or indoor values of air temperature (r = 0.233; p > 0.05), relative humidity (r = −0.095; p > 0.05) and airflow rate (r = 0.216; p > 0.05), indicating controlled production conditions. Both indoor and outdoor mean relative humidity was higher in winter (Tables 1 and 2, respectively); however, unlike outdoor relative humidity, indoor relative humidity in winter increased with fattening weeks. This could be due to reduced ventilation in winter to prevent heat losses and avoid additional heating costs. That is why the concentrations of gaseous air pollutants in poultry housing usually are higher in winter months as well [30,31], as also confirmed by the results of this study. Ammonia concentration in house air generally increased significantly (p < 0.05) during fattening period in both seasons, except for a decrease in the last fattening week in winter. Comparison of the two seasons showed higher ammonia concentrations, with significantly higher values (p < 0.05) in the third and fourth fattening weeks and a significantly higher (p < 0.001) mean ammonia concentration during fattening in winter ( Table 1).
The concentrations of airborne fungi in broiler houses commonly range from 10 3 to 10 5 CFU/m 3 , with Aspergillus, Penicillium, Cladosporium, Alternaria, Fusarium and Scopulariopsis as predominant fungal genera. The following genera also are quite commonly found: Rhizophus, Mucor and Geotrichum [21,23,27,32]. In our study, total fungal count in broiler house air was consistent with previous research, having increased significantly (p < 0.05) by the mid-fattening period in both seasons (Figure 1), which could be explained by higher broiler activity in the initial fattening period, whereafter their activity decreased due to greater body mass and reduced mobility, thus decreasing the level of air pollution [27]. With the exception of the fourth week, total fungal count in the house air was significantly higher (p < 0.05) throughout the fattening period in summer as compared with winter (Figure 1), and so was the mean total fungal count (p < 0.01) in the house air during summer (Table 3). Although a significant positive correlation was found between fungal count in the air and relative humidity inside house (r = 0.437; p < 0.05) ( Table 4), the results obtained could be explained by the lower mean indoor humidity in summer (Table 1). It is known that dust particles serve as microorganism carriers, and dust concentrations in poultry housing are higher at lower humidity [19,33]. Lawniczek-Walczyk et al. [21] did not find significant correlation between air humidity and fungal count in broiler housing or seasonal differences in fungal count, whereas Wójcik et al. [23] report on a higher airborne fungal count in winter as compared with summer. However, in their study, relative humidity was lower in winter, suggesting that fungal contamination should not be associated exclusively with season but rather with the real house microclimate. Contrary to the findings of Viegas et al. [19] and Popescu et al. [20], there was no significant correlation of fungal count in house air with indoor air temperature and airflow rate (Table 4); yet, in line with our report, the latter authors [20] found significant positive correlation between relative humidity and fungal count in poultry house air.
Investigating the effect of environmental conditions on mycoflora in poultry housing, Debey et al. [22] found the air to contain higher Aspergillus sp. concentration in winter as compared with summer, which was associated with a higher dust level and lower relative humidity, indicating that the spores of these fungi were more frequently found in dry than in humid air. This was supported by the results of our study. Although A. fumigatus and A. terreus were not isolated in the house air or broiler trachea in summer, Aspergillus sp. including A. flavus and A. niger was significantly more frequently detected (p < 0.001) both in the house air and broiler trachea in summer (Tables 5 and 6). However, in summer, the mean indoor relative humidity was lower as compared with winter. On the other hand, Mucor sp. and Penicillium sp. were significantly more commonly detected (at least at p < 0.05) in house air and broiler trachea in winter. In addition, Cladosporium sp. was more frequently isolated in the air and broiler trachea in summer, although its count in broiler trachea showed significant seasonal difference (p < 0.001). Although identified in house air and broiler trachea in both seasons, only the concentrations of Fusarium sp. and Rhizopus sp. did not yield seasonal differences. Thus, six mould genera including four Aspergillus species and Trichophyton sp. isolated in broiler trachea in winter were isolated in house air and broiler trachea in both seasons (Tables 5 and 6).
On average, yeasts predominated in house air and broiler trachea in both seasons, being significantly higher in house air in summer (p < 0.001), and in broiler trachea in winter (p < 0.01) (Tables 5 and 6), confirming association between the fungal flora composition in house air and broiler trachea, as well as the seasonal effect of fungal composition. In addition, these findings suggest that yeasts contribute significantly to total fungal count. Thus, the average total fungal count was higher in house air in summer and in broiler trachea in winter, although a significant positive correlation was found between fungal count in house air and in broiler trachea (r = 0.208; p < 0.05) ( Table 4). Debey et al. [22] found yeast count to be higher in poultry house air in winter, which could be due to lower relative humidity, as previously discussed. Accordingly, higher airborne yeast concentration in our study in summer could be explained by lower mean relative humidity inside the house found in this season.
In summer, fungal count in broiler trachea decreased significantly (p < 0.05) after the second fattening week and maintained the level achieved until the end of the fattening period, whereas in winter it increased significantly (p < 0.05) (Figure 2). Accordingly, in spite of the lower fungal count in house air in winter, fungal count in broiler trachea was higher as compared with summer. These results could be explained by the significant positive correlation (r = 0.691; p < 0.05) between relative humidity inside house and fungal count in broiler trachea (Table 4), i.e., by the higher mean indoor relative humidity in winter, whereby air humidity favoured deposition of fungi and tracheal infestation. Thus, more fungal genera were detected in broiler trachea in winter than in summer (Table 6). Besides this, ammonia concentration in house air was higher in winter than in summer. Ammonia has been demonstrated to be one of the main precursors of secondary particles [30,34]. Kumari et al. [35] found a significant positive correlation of air ammonia and secondary particles with fungal abundance. In our study, a significant positive correlation was also recorded between ammonia concentration and fungal count both in house air (r = 0.335; p < 0.05) and broiler trachea (r = 0.491; p < 0.05) ( Table 4). In addition, tracheal fungal count was significantly correlated with air temperature (r = −0.437; p < 0.05) and airflow rate in the broiler house (r = 0.197; p < 0.05) ( Table 4).
Potentially harmful fungi were identified in both seasons. Aspergillus flavus and A. niger are known to produce mycotoxins and can have a role in the outbreak of aspergillosis, A. flavus in particular [16,36]. Although Aspergillus sp., predominantly A. flavus, prevailed both in house air and in broiler trachea in summer, A. fumigatus and A. terreus were found in house air and in broiler trachea in winter. Also, Trichophyton sp. was isolated from broiler trachea in winter. According to the Commission Directive (EU) 2019/1833 [37], Aspergillus sp. and Trichophyton sp. are classified into group 2 biological agents considering the risk of infection in occupational environment. Penicillium sp., also known as a mycotoxin producer [36], was also more frequently identified in broiler trachea, as well as in house air in winter. Fusarium sp. is another mycotoxin producing fungus [36], but there were no seasonal differences in its concentrations either in house air or in broiler trachea. In addition, Fusarium sp. has been reported among the emerging causes of opportunistic mycoses in humans and animals [38,39].

Conclusions
The results of our study revealed a seasonal impact on fungal count and composition in poultry house air and in broiler trachea, demonstrating the association of fungal flora in house air and in broiler trachea. Indoor relative humidity and ammonia concentration were found to influence total fungal count in both poultry house air and broiler trachea; yet, in order to obtain exact results on seasonal effects, fungal determination should be performed both in the birds and in their environment. Nonsignificant correlation between outdoor and indoor air temperature, relative humidity and airflow rate indicated that seasonal differences in fungal contamination should not be related to season alone but rather to the housing microclimate conditions. The fungi detected in the study pose a health risk for both the poultry and the humans working in poultry environment.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/atmos12040459/s1, Figure S1: Air temperature inside broiler house according to fattening weeks in summer and winter, Figure S2: Relative humidity inside broiler house according to fattening weeks in summer and winter, Figure S3: Airflow rate inside broiler house according to fattening weeks in summer and winter, Figure S4: Air ammonia concentration in broiler house according to fattening weeks in summer and winter, Figure S5: Air temperature outside broiler house according to fattening weeks in summer and winter, Figure S6: Relative humidity outside broiler house according to fattening weeks in summer and winter, Figure S7: Airflow rate outside broiler house according to fattening weeks in summer and winter.

Conflicts of Interest:
The authors declare no conflict of interest.