Characterisation of the Mycobiota on the Shell Surface of Table Eggs Acquired from Different Egg-Laying Hen Breeding Systems

Microbial safety is an important factor contributing to the egg quality. During egg acquisition, there is significant risk of contamination of the eggshell surface with microscopic fungi. Mycelial hyphae may grow on the eggshell surface and penetrate into the egg content. However, there is no information on the populations of microscopic fungi on the eggshell surface and, consequently, on possible production of mycotoxins. Therefore, the aim of the study was to identify the species of microscopic fungi present on the eggshell surface acquired from different breeding systems and to measure the number of selected mycotoxins. The qualitative analysis resulted in the identification of 41 isolates on the surface of eggs. There were 7 isolates from the organic production system, 11 from the free-range production system, 14 from the deep litter indoor housing system and 9 from the cage farming production system. The research proved that the diversification in the population of mycobiota on the eggshells depended on the egg-laying hen breeding system. The microscopic fungi isolated from the eggshells included toxigenic and pathogenic species such as Fusarium culmorum and F. equiseti. As the egg storage time increased, fungi, including the pathogenic species, penetrated through the eggshells. In consequence, mycotoxins were identified in the egg whites. Type-A and type-B trichothecenes were found in the eggshell samples containing F. culmorum.


Introduction
Eggs are important food all over the world. Following China, the European Union is the second largest producer of eggs for consumption-its share in the global production amounts to about 10% [1]. Due to the high consumption of eggs it is important to guarantee high standards of consumer safety during production process. Therefore, scientists are conducting research to identify potential risks to consumers' health. Most of their attention is concentrated on the bacterial microflora [2][3][4]. However, latest studies point out a threat caused by the eggshell contamination with microscopic fungi [5]. So far, the problem of mycobiota has been studied on hatching eggs. The conditions of incubation (high air humidity and diffusion of vapour from egg content) favour the development of microscopic fungi on eggshells. In consequence, embryos become contaminated with mycelium and they die [6,7]. Due to these risks, also table eggs were subjected to investigations. It was already proven that the shells of table eggs become contaminated with fungi and that mycelial hyphae penetrate through eggshell membranes [8].
Eggshell contamination may be initiated due to the conditions of egg acquisition, that is, the quality of straw and feed, the presence of dust, temperature and moisture. We can suppose that the eggs produced in the deep litter indoor housing, free-range or even organic systems are at the highest risk of contamination with microscopic fungi. In the deep litter indoor housing system, the contamination is usually caused by moist litter or dirty nests. Researchers studying different types of litter found that it contained fungi from Aspergillus, Cladosporium, Drechslera, Penicillium, Stemphylium and Fusarium genera [9][10][11]. Straw chaff was found to be the most contaminated as its CFU/g was the highest (28.49 log CFU/g). It also displayed the highest concentration of ergosterol (ERG) (605.74 mg/kg), which is a chemical indicator of material contamination with microscopic fungi [11]. Fungi not only degrade the substrate on which they occur but they also cause numerous diseases due to the presence of spores in the air [12].
Mycotoxins, which are secondary metabolites produced by some fungal strains, are the greatest risk caused by microscopic fungi, particularly in feeds used for breeding farm animals [13]. The content of mycotoxins in more than a half of the farm animal feeds samples collected in 2010 exceeded the maximum legal limit. In Europe, the feed samples usually contained deoxynivalenol (DON) and T-2 toxin, which belong to the trichothecenes, as well as zearalenone (ZON). The feed samples from Asia and the Pacific region were usually contaminated with DON, ZON, fumonisins and aflatoxins [14]. Mycotoxins from the trichothecene group, fumonisins and zearalenone belong to the most toxic metabolites produced by the Fusarium species and were proven to be the most harmful when consumed [15]. Due to the increasing number of reports on the contamination of food products and ingredients with microscopic fungi, it seems interesting to investigate their populations on eggshells and their potential to produce mycotoxins.
The aim of the study was to conduct molecular identification of the species found in mycobiota present on the shell surface of table eggs acquired from different egg-laying hen breeding systems and to identify and quantify the mycotoxins in the respective egg white samples.

Mycobiota Species Identification
Eggs selected for analyses were stored at programmed humidity and temperature, which were similar to the conditions in warehouses and shops. The analyses were conducted on eggs laid by hens kept in 4 systems and in 2 experimental variants, that is, with and without sterilisation (control variant). Surface sterilisation effectively eliminates microorganisms adhering to the eggshell surface. The procedure is applied to acquire isolates from eggshell pores. The qualitative analysis resulted in the identification of 41 isolates on the surface of eggs. In total, there were 7 isolates from the organic production system, 11 from the free-range production system, 14 from the deep litter indoor housing system and 9 from the cage farming production system.
Fungi belonging to the Alternaria, Penicillium and Chaetomium genera were the most frequently isolated from the eggshell surface ( Figure 1). Alternaria species isolated in the experiment chiefly colonise crops, including cereal grains [16]. These fungi have the potential to generate toxins such as alternariol and tenuazonic acid. It was found that the Penicillium sp. isolated in the experiment accelerated the egg rotting process and caused qualitative changes in the egg content (results not shown). Chaetomium sp., which was isolated from the eggshells, is a fungal species commonly found in soil, straw, cereal grains and on wet surfaces of buildings all over the World [17][18][19].

Mycobiota Strains Isolated from Eggs Acquired from Different Egg-Laying Hen Breeding System
This research showed that composition of fungal species isolated from the eggshells differed according to the egg-laying hen housing system (Table 1). There were qualitative and quantitative differences in the fungal species between the sterilised eggs and the control samples (without sterilisation). The highest species diversity was noted in the population of fungi isolated from eggs laid by hens kept in the deep litter indoor housing system. Samples originating from this system mostly contained fungi of the Alternaria genus (5 species). There were also two fungal species of the Fusarium genus isolated. So far, no reports became available on the occurrence of these species on the eggshell surface. The hygienisation procedure enabled the isolation of Scopulariopsis brevicaulis, Trichothecium roseum and Acrostalagmus luteoalbus species from the shell surface of eggs laid by hens kept in the deep litter indoor housing system. These species were not observed on the shell surface of eggs laid by hens kept in the other housing systems. The diversity of potentially pathogenic fungal species on the eggshell surface is related to the unique microclimate inside the henhouse with the deep litter system, that is, high air humidity and temperature, poor ventilation, exogenous contamination, (litter, feed) and endogenous contamination (dust) [5].

Mycobiota Strains Isolated from Eggs Acquired from Different Egg-Laying Hen Breeding System
This research showed that composition of fungal species isolated from the eggshells differed according to the egg-laying hen housing system (Table 1). There were qualitative and quantitative differences in the fungal species between the sterilised eggs and the control samples (without sterilisation). The highest species diversity was noted in the population of fungi isolated from eggs laid by hens kept in the deep litter indoor housing system. Samples originating from this system mostly contained fungi of the Alternaria genus (5 species). There were also two fungal species of the Fusarium genus isolated. So far, no reports became available on the occurrence of these species on the eggshell surface. The hygienisation procedure enabled the isolation of Scopulariopsis brevicaulis, Trichothecium roseum and Acrostalagmus luteoalbus species from the shell surface of eggs laid by hens kept in the deep litter indoor housing system. These species were not observed on the shell surface of eggs laid by hens kept in the other housing systems. The diversity of potentially pathogenic fungal species on the eggshell surface is related to the unique microclimate inside the henhouse with the deep litter system, that is, high air humidity and temperature, poor ventilation, exogenous contamination, (litter, feed) and endogenous contamination (dust) [5].
There was similar diversity of fungal species observed in the eggs laid by hens kept in the free-range system. Similarly, to the eggs from the deep litter system, there were also pathogenic species of Alternaria and Fusarium isolated on the eggshell surface ( Table 1). The possibility to control hen breeding conditions in the free-range system is limited. As hens have a free access to full-value feed or green forage, litter and the range, there is higher risk of contact with pathogenic organisms of different origin [20].
There were ten fungal species observed on the shell surface of eggs laid by hens kept in the organic system with no pathogenic species of the Fusarium genus among them ( Table 1). The hygienisation procedure enabled the isolation of a large population of fungal species in this system. Most of the species were characteristic for the henhouse environment. In the organic system, a limited number of hens is kept per square meter. Consequently, effective control of the henhouse climate is possible. Adequate humidity and temperature reduce the risk of the extensive growth of pathogenic fungi. There were eight fungal species isolated on the shell surface of eggs laid by hens kept in the cage system. Only two of them had the potential to produce mycotoxins ( Table 1). The sterilisation procedure allowed for the isolation of the Engodontium album species on the eggshell surface. This species was also isolated from the eggs with the mycoflora native to the deep litter system. Although E. album has not been reported to be mycotoxigenic, its spores and mycelium can cause infections in humans causing brain abscess and keratitis [21]. In the present study, the conditions in a henhouse with the cage system resulted in low fungal diversity on the eggshell surface. The design of furnished cages allowed for the natural preferences of hens and the safety of eggs produced [22,23]. As far as the microbial safety of eggs is concerned, the advantage of the cage system results from the fact that it does not use straw, which is a potential vector of pathogenic fungal species from the field.
In conclusion, the qualitative analysis of the fungi isolated showed that the shell of table eggs was a potential substrate for the growth of numerous fungi, including pathogenic and toxin-producing species, for example, Fusarium and Alternaria. Moreover, the diversity of the fungal population differed according to the egg-laying hen housing system. The fungal species present on the eggshell surface may occur in the environment of the henhouse [9].

Toxin Content Analysis
The presence of Fusarium spp. fungi on the eggshell surface involves the potential risk of their presence and production of mycotoxins in the egg content. The content of type-A and type-B trichothecenes was measured in order to check the potential contamination of eggs with mycotoxins (Tables 2 and 3). Only the egg white was analysed for the content of mycotoxins. Szablewski et al. [8] proved that there were no fungi in the yolk after two weeks of storage. There were no Fusarium mycotoxins found in freshly laid eggs. This means that no Fusarium mycotoxins were transmitted from the feed through the alimentary tract into the eggs. There were type-A and type-B trichothecenes in the content of the eggs whose shell samples contained F. culmorum. This species chiefly synthesizes DON, 3-AcDON and NIV [15,24]. The content of type-A trichothecenes may indicate the presence of species such as F. sporotrichioides [25,26]. We did not identify any Fusarium species producing type-A trichothecenes in our study. It may have been caused by the fact that only viable fungal species growing on the eggshell surface were analysed. After the period of egg storage, the pathogenic species of microscopic fungi may have penetrated into the egg content. They may have died because of shortage of nutrients or competitive microflora on the eggshell surface. Table 2. Mean values and standard deviations of type-A trichothecenes present in egg samples contaminated with Fusarium species.
The opposite situation was observed in some samples. They contained the viable mycelia of microscopic fungi which potentially produce mycotoxins but no mycotoxins were found in the samples. This finding is not surprising, as the biosynthesis of these metabolites may be modified by the occurrence of abiotic or biotic stress factors. For example, under specific conditions the F. equseti species is capable of producing selected trichothecene mycotoxins and zearalenone [25,27,28]. Unfortunately, no mycotoxins produced by F. equseti were analysed in our study.

Toxin Content Analysis in Eggs Acquired from Different Egg-Laying Hen Breeding System
Our study did not prove any correlation between the content of mycotoxins in egg white and the egg-laying hen breeding system ( Table 4). The content of mycotoxins in egg white from hens bred in the organic and cage breeding systems was below the detectable level. There were no significant differences in the concentrations of mycotoxins in egg white from hens bred in the deep litter and free-range systems. Until recently, the issue of the egg content contamination with mycotoxins produced by pathogenic species growing on the egg surface and in the egg content has not been scientifically investigated. So far, the experiments were focused on the transmission of mycotoxins from feed through the alimentary tract into the egg content [29][30][31][32]. In the present study, we have shown that the level of mycotoxins in eggs is not harmful to the human body. However, attention should be paid to the possibility of the bioaccumulation of mycotoxins in the human body [33]. Our study reports the new potential source of chemical and microbial risks in the production of table eggs. <LOD a -no differences at the level of significance α = 0.05.

Conclusions
The research showed that the diversity of the mycobiota population on the eggshell surface depended on the conditions of maintenance of egg-laying hens. The microscopic fungi isolated from the eggshells included toxigenic and pathogenic species such as F. culmorum and F. equiseti. As a consequence, there were mycotoxins present in the egg whites. The production of mycotoxins in the egg content may indicate that microscopic fungi competed with other microorganisms. The results showed that the conditions of the henhouse environment significantly influenced the initial eggshell contamination. These main sources of contamination should be taken into consideration when preparing strategies preventing the occurrence of toxin-producing fungal species in table eggs.

Egg Collection
The research was conducted on 240 eggs (class M, average weight 53 g) acquired from hens kept in the cage farming production system (Hy-Line White), deep litter indoor housing system (Hy-Line), free-range production system (Green-Legged Hens) and organic production system (Green-Legged Hens) with the 'PL-Eko' certificate. The henhouses were located in central Poland.

Mycobiota Isolation, Media and Culture Conditions
On being laid, half of all egg samples were subjected to surface sterilisation in an aqueous ethanol (70%) solution for 30 seconds. All the eggs were stored at high humidity (95%) and temperature (20-25 • C) in a climate chamber (Binder KBF 115,Tuttlingen,Germany). After two weeks of storage the eggs were broken and divided into fractions. Shredded eggshells were aseptically cultured on Potato Dextrose Agar (PDA, Oxoid, Basingstoke, UK). Eggshell mycobiota were incubated at 20-25 • C for 5-7 days. The purification of cultures was conducted according to the manual [34]. Pure fungal cultures were grown on the PDA medium until the genomic DNA extraction.

DNA Extraction
Forty-one fungal cultures were isolated and purified from eggshells. The following molecular techniques were used in the pre-validated procedure. Mycelia of individual isolates were harvested from the PDA plates after 7 days of incubation. Genomic DNA was extracted using the hexadecyltrimethylammonium bromide (CTAB) method used previously [35]. DNA extracts were stored at −20 • C.
For sequence analysis, PCR-amplified DNA fragments were purified with exonuclease I (Thermo Scientific, Espoo, Finland) and shrimp alkaline phosphatase (Thermo Scientific, Espoo, Finland) using the following programme: 30 min at 37 • C and 15 min at 80 • C. DNA fragments were labelled using forward primer and the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to the method described in Stępień et al. [39] and precipitated with 96% ethanol. Sequences were read using Applied Biosystems equipment (Applied Biosystems, Foster City, CA, USA). The sequences were aligned using BLASTn algorithm to the sequences of reference strains belonging to individual fungal species, deposited in the GenBank Database (National Center for Biotechnology Information, Bethesda, MD, USA). They were assigned to the reference species, where both sequence coverages and nucleotide identities matched the query at 99-100%.