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Differences in Fungal Contamination of Broiler Litter between Summer and Winter Fattening Periods

Abstract This study aimed to compare fungal contamination of poultry litter between warm and cold seasons. It was carried out in commercial production conditions over two five-week fattening periods: one in the summer (July-August) and one in the winter (December-January). Broilers were reared on a litter composed of chopped straw and sawdust. Litter fungal concentration and composition were investigated weekly, along with litter temperature, moisture, and pH. Litter concentration of total fungi increased over both fattening periods, with no differences in median concentrations between them. Season also had no effect on yeast, Aspergillus section Nigri, and Cladosporium, Fusarium, and Rhizopus spp. concentrations, while the Aspergillus section Flavi and Aspergillus spp. combined showed higher concentrations in the summer, and Mucor and Penicillium spp. in the winter. Total fungal concentration highly correlated with litter temperature, moisture, and pH, regardless of the season. Our findings can be useful in the assessment and control of potential harmful effect of fungi on the health of poultry and poultry farm workers.

The aim of our study was to get a broader picture of fungal contamination (than limiting ourselves to Aspergillus spp.) by investigating seasonal fungal flora in poultry litter and test the following hypotheses: (i) the season will influence total fungal concentrations and composition in the litter, and (ii) fungal concentrations will depend on other parameters of litter quality, such as temperature, moisture, and pH.

MATERIALS AND METHODS
The study protocol was approved by the Ethics Committee in Veterinary

Study design, animals, and farm management
The study was conducted in commercial farming conditions in a broiler house over the five-week production cycles in the summer (July-August 2016) and winter (December-January 2016/2017). During each production cycle, 18,000 Ross hybrid broilers were kept in controlled conditions on a 10-cm thick litter made of a 2-cm bottom pine sawdust layer and a 8-cm wheat straw layer (up to 33 kg/m 2 stocking density). The broiler house used straw from its own farming fields (chopped to the length of 5 cm) and sawdust from a sawmill (neither dedusted nor disinfected). Litter was neither turned over nor additional amount of litter was added during either production cycle. The broiler house has an oil heater, negative pressure mechanical ventilation, and lighting as recommended for Ross hybrids (33). Broilers were fed complete feed mix (Biodar, Varaždin, Croatia) from round pan feeders and watered from nipple drinkers with cups. Feed and water were provided ad libitum.
The broiler house is cleaned and disinfected with highpressure cleaners between production cycles, which are separated by a two-week house rest. Floors are disinfected with caustic soda (PCC Rokita SA, Brzeg Dolny, Poland) and the rest of the house, including equipment, with Ecocid ® S (Krka d.d., Novo Mesto, Slovenia). Once the litter is spread, the house is fumigated with a Formaster G tablet (Formaster di Emanuela Magnani & C.s.a.s., Piacenza, Italy).
Litter fungal contamination, temperature, moisture, and pH were determined once a week over the five-week production cycles.

Litter sampling and analytical methods
Litter temperature (°C) was measured with a Testo 925 thermocouple thermometer (Testo SE & Co. KGaA, Lenzkirch, Germany) at 3 cm below litter surface at five points of the broiler house (four corners and one in the centre). At these points we also took one litter sample per point into sterile bags (Aptaca Spa, Canelli, Italy) and transported them to the laboratory for analysis on the same day. Litter moisture (%) was determined using the gravimetric method by calculating weight loss after drying, whereas pH was determined electrometrically using a WTW inoLab pH 720 pH meter (Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany). More detailed descriptions are available elsewhere (8,34).
Litter fungal contamination was determined as follows: 10 g of each sample was first diluted 10-fold with sterile distilled water and then 100 µL of the dilution plated on Sabouraud dextrose agar (Biolife, Milan, Italy) and incubated at 25 °C for 5-7 days. The fungi were identified based on macroscopic appearance of grown colonies and on microscopic examination of spores stained with lactophenol blue solution (Sigma-Aldrich, St. Louis, MO, USA) (35,36). The concentration was expressed as colony forming units per gram of litter (CFU/g). Analytical procedures were performed in triplicate.

Statistical analysis
All data were analysed with Statistica v. 13.5 (TIBCO Software Inc., Palo Alto, CA, USA, 2018). The normality of data distribution was tested with the Kolmogorov-Smirnov test. The significance of differences in median litter fungal concentrations between summer and winter measurements was tested with the Mann-Whitney U-test. Differences in mean litter temperature, moisture, and pH and differences between weekly measurements of all parameters between the seasons were compared with Student's t-test. Differences between weekly measurements within one season were tested with the repeated-measures ANOVA and post-hoc Tukey HSD test. Correlations between fungal concentrations and litter temperature, moisture, and pH were assessed with Spearman rank-order correlation. In all tests, the level of statistical significance was set at P<0.05.

RESULTS AND DISCUSSION
Litter fungal concentrations ranged from 10 2 to 10 5 CFU/g, which is consistent with previous reports (9,21,(37)(38)(39), and increased with time (fattening weeks), as expected (39). We found no significant seasonal differences at the beginning and the end of the fattening periods ( Figure  1) or in the median fungal concentrations for either period (Table 1). This is because fungal concentrations highly correlated with litter moisture and pH throughout the study (Table 2), which did not significantly differ between the seasons ( Table 1). The correlation between fungal growth and litter moisture and pH was also reported by Hutson (40) and Schefferle (41), but there are reports to the contrary as well (38). According to Arné et al. (5), the impact of variations in litter moisture and pH on fungal population density remains a controversial issue, although wet and soiled areas can intensify fungal growth.
Even though the five-week fattening periods in our study were shorter than in the above studies, and higher litter moisture and pH were recorded in the last week of both periods, the comparison makes sense, as according to Milanov et al. (25) microorganism growth (including fungal) in broiler litter reaches its peak in about one month of fattening, after which it declines and reaches steady levels.
We also found a highly significant correlation between fungal concentrations and litter temperature (Table 2), which increased with time in both seasons (Figure 2), yielding values consistent with the reports by Spindler and Hartung (42) and Knížatová et al. (43). However, the latter study found no major differences in litter temperature between summer and winter fattening periods, whereas we did (Table 1, Figure 2). Despite controlled conditions in the broiler house, this was likely related to seasonal differences in outdoor temperature. Even so, it seems that the five-week fattening periods in our study were too short for the differences in litter temperature to cause seasonal differences in fungal concentrations or to affect litter moisture and/or pH.
Litter moisture and pH in our study increased over both seasons as weeks went by (Figures 3 and 4) and were consistent with other studies of straw as a litter material (44)(45)(46). Their mean values did not significantly differ between the seasons (Table 1), save for the higher moisture level in the last fattening week of the winter fattening    Figure 3). Comparing different materials for broiler litter between summer and winter fattening periods, Garcia et al. (47) reported higher moisture for straw in the summer and higher pH in the winter, which may be owed to different farm management in their study. Previous studies have revealed that litter moisture also depends on litter quantity, stocking density, watering system, ventilation, indoor microclimate, broiler age, nutrition, and health (enteritis) (48,49). Even though we did not keep track of broiler health, no enteritis outbreaks were recorded in either season.
Our findings have shown a prevalence of yeasts in broiler litter, with no significant seasonal difference (Table  3), as also reported by other studies (23,25,26,37).
Besides yeasts, the most prevalent fungal species detected in our study were Aspergillus (encompassing the Flavi and Nigri sections), Mucor, and Penicillium spp. (Table 3), which is in line with previous reports (9, 22, 37-39, 50, 51). These three also showed significant seasonal differences. Aspergillus spp. combined, including A. section Flavi, were more frequent in the summer, and Mucor and Penicillium spp. in the winter. Other species showed no seasonal differences (Table 3). Soliman et al. (23) also found Aspergillus spp. to prevail in closed broiler houses in the summer and Penicillium spp. in the winter, but unlike our study, theirs reported Mucor spp. to be more prevalent in the summer.
Some studies using same methodology for fungal identification detected more mould genera, including Aspergillus sections, than we did (9,37,38), most likely because of different litter material. This suggests that straw litter, known to have higher moisture, pH, and temperature than other materials (44,(52)(53)(54), favours the development of more homogeneous mycoflora.
Even though we used the same methods for fungal identification as Chollom et al. (24), we did not find any dermatophytes in the litter, which are of particular health concern. Their finding of high Trichophyton spp. prevalence in poultry litter may be owed to higher incubation temperature than in our study, but may also point to our less precise fungal identification.
Nevertheless, several fungal genera isolated in our study pose a health risk both as pathogens or producers of mycotoxins, which can cause severe poisoning in humans and animals (55), Aspergillus spp. in particular.
Aspergillus fumigatus (section Fumigati) is the most pathogenic fungus affecting poultry, accounting for 95 % of all cases of aspergillosis, but other species, such as A. flavus (section Flavi), A. niger (section Nigri), A. nidulans (section Nidulantes), and A. terreus (section Terrei), alone or combined, also present a health risk (14,56,57). Besides birds, Aspergillus species are known opportunistic pathogens in humans, immunocompromised patients in particular. Aspergillus fumigatus accounts for over 80 % of diseases, including invasive pulmonary aspergillosis, aspergilloma, and different forms of hypersensitivity disorders such as allergic asthma, allergic sinusitis, pneumonitis, and allergic bronchopulmonary aspergillosis (58)(59)(60). Another species with emerging evidence of a broad spectrum of infections that are difficult to treat are the Fusarium spp., which can cause onychomycosis, skin infections, and keratitis (61). We did not find any species from the Aspergillus section Fumigati in our litter samples, regardless of the season, while Fusarium spp. was detected in the winter ( Table 3).
As regards the risk of mycotoxin poisoning, A. sections Flavi and Nigri, and Fusarium and Penicillium spp. we

CONCLUSIONS
Our findings suggest that season does not influence total fungal concentrations as much as fungal composition in broiler litter. They have also confirmed that fungal concentrations much depend on litter temperature, moisture, and pH and can be useful in the assessment and control of potential adverse effects on poultry and poultry farm workers.
To the best of our knowledge, other studies did not compare fungal contamination of broiler litter on the same farm between seasons. Furthermore, our study contributes to the scarce data available on straw as litter material in broiler rearing. What limits the interpretation of our findings is that they do not distinguish enough fungal isolates at the species level, as we used methods specific enough to allow comparison with the results of other studies investigating poultry litter mycoflora. Future studies should observe various litter materials in the same production conditions across seasons with more specific identification of fungi, including the use of sensitive methods of genotyping.

Conflicts of interest
None to declare.