Geophilic dermatophytes and other keratinophilic fungi in the nests of wetland birds

The frequency and species diversity of keratinophilic fungi in 38 nests of nine species of wetland birds were examined. nine species of geophilic dermatophytes and 13 Chrysosporium species were recorded. Ch. keratinophilum, which together with its teleomorph (Aphanoascus fulvescens) represented 53% of the keratinolytic mycobiota of the nests, was the most frequently observed species. Chrysosporium tropicum, Trichophyton terrestre and Microsporum gypseum populations were less widespread. The distribution of individual populations was not uniform and depended on physical and chemical properties of the nests (humidity, pH).


INTRODUCTION
Geophilic dermatophytes and species representing the Chrysosporium group (an arbitrary term) related to them are ecologically classified as keratinophilic fungi.Keratinophilic fungi colonise keratin matter (feathers, hair, etc., animal remains) in the soil, on soil surface and in other natural environments.They are keratinolytic fungi physiologically specialised in decomposing native keratin.They fully solubilise native keratin (chicken feathers) used as the only source of carbon and energy in liquid cultures after 70 to 126 days of growth (20°C) (Korniłłowicz-Kowalska 1997).Fungi other than dermatophytes and Chrysosporium decompose only 30%-33% of native feather keratin in the same period (Korniłłowicz-Kowalska l.c.).According to Kunert (2000), fungi are weakly keratinolytic if they decompose no more than 40% of keratin in liquid cultures after eight weeks and non-keratinolytic if they decompose less than 20%.native keratin substrates contain both keratin as well as simpler compounds, e.g., non-keratin proteins, amino acids, urea, which constitute up to 10% of the substrate's dry weight (Mercer 1958).This allows a range of other fungi that are de facto non-keratinolytic to grow on native keratin (Korniłłowicz 1992).
Ubiquistic moulds produce only thin and simple boring hyphae when growing on native keratin substrates (English 1965).
The process of fungal keratinolysis consists of three stages: deamination, sulphitolysis and proteolysis (Kunert 2000).Deamination leads to the release of ammonia conditioned by a high nitrogen level in native keratin: from 14.72% in feathers (Korniłłowicz-Kowalska 1997) to 16% in hair (Kunert 2000), and a narrow C:n ratio in these substrates, such as 3:1 for feathers (Korniłłowicz-Kowalska l.c.).n-nH 4 + accumulation causes environment alkalisation necessary for enzymatic disruption of greatly numerous keratin disulphide bridges responsible for its resistance to the activity of proteolytic enzymes.Sulphitolysis, that is the process of the disruption of S-S bonds, occurs with the participation of inorganic sulphite produced by the fungus (Kunert 1973(Kunert , 1976)).This leads to keratin denaturation and, consequently, makes proteolysis with alkaline or neutral proteases of these fungi possible (Kunert 2000).
During saprotrophic growth on native keratin, keratinolytic fungi oxidize 70% of carbon to Co 2 , release 70-80% of nitrogen as ammonia and transform 30-50% of sulphur into sulphates (Korniłłowicz-Kowalska 1997).This allows keratinolytic fungi to play an important role in the recycling of carbon, nitrogen, sulphur of animal remains containing keratin.
The occurrence of keratinophilic fungi in natural environments is conditioned primarily by their "animialisation" related to an inflow of keratin matter (Montovani et al. 1982).Keratin remnants are not only a nutritive source for the fungi but also a specific habitat enabling their survival and defence from other competitive microorganisms (Garetta, Piontelli 1975).The species diversity of keratinomycetes also depends on various physical and chemical properties of the environment, mostly pH, humidity and temperature (Bőhme, ziegler 1969;Chmel et al. 1972;Chmel, Vláčiliková 1975;Garg et al. 1985;Kushwaha 2000;Korniłłowicz-Kowalska, Bohacz 2002).
As the participation of wetland birds in the distribution of various pathogenic micro-organisms, including opportunistic pathogens causing mycoses in birds, mammals and humans has been on the increase in recent years (Hubalek 2004), there is a need to expand studies on the occurrence and distribution of keratinophilic fungi in breeding and feeding biotopes of these birds.Such research will contribute to a better knowledge of the role of these habitats in the survival and, partly, transmission of potentially pathogenic keratinophilic fungi.
The aim of this study was to identify the species composition and the frequency of geophilic dermatophytes and Chrysosporium representatives in the nests of different species of wetland birds in connection with some physical and chemical properties of those nests.

MATERIAL AnD METHoDS
Nests: location, building material and structure.nests of nine bird species in southeast Poland (the Lublin region) were examined.A total of 38 nests were studied.The nests were collected in the period between 2006 and 2008 after they had been abandoned by birds.only nests of grey herons (Ardea cinerea) were situated in the trees; the nests of other birds were in reeds and rushes of water bodies (ponds, a lake) in other aquatic vegetation or were floating nests (Tab.1).
The nests were built from plant material and contained various amounts of nonplant material which was partly used to line the nest (feathers, hair) and was partly secondary (shed feathers, food remains, etc.) (Tab.2).
Nests of marsh harriers (Circus aeruginosus) were 50-90 cm in diameter and were partly above the water surface (ca.50 cm under water and ca.70 cm above water).The nests were stable, non-floating, situated in reed beds of the common reed (Phragmites australis) or rushes of the broadleaf cattail (Typha latifolia).The base of the nests was built from twigs of black alder (Alnus glutinosa), downy birch (Betula pubescens) and other birches (Betula sp.) as well as willows (Salix sp.).nest edges were sometimes supported with stems of burdock (Arctium sp.) and creeping thistle (Cirsium arvense).The lining, easily distinguishable from the rest of the nest, was built from common reeds and broadleaf cattails, supplemented with sedges Carex sp. and Poacae grasses difficult to identify due to rotting.The lining also contained great nettle (Urtica dioica), great bulrush (Schoenoplectus lacustris), sometimes rhizomes of weed grass Agropyron sp. and other unidentified small roots.Material of animal origin constituted much of the lining: pellets of adult and young birds containing mammal hair, bird feathers, other undigested parts of the prey and prey bones; remains of uneaten prey containing hair of small mammals Micromammalia, Both plant and non-plant components of the nests' building material were highly compressed because of the birds' presence in the nests and formed a fixed structure.
Nests of grey herons (Ardea cinerea) were in a heronry in the crowns of tall trees.The base of the nests (a loose cone) was built from thicker sticks; the inner part was built from flexible twigs of deciduous trees: birch (Betula sp.), willow (Salix sp.) and alder (Alnus sp.).The nest lining was sparse, mostly composed of grasses (Poacae), various unidentified small roots as well as hair, feathers and fish scales.The lining was absent in some nests.The nest structure was covered with a high amount of white excrements on the inside and the outside as chicks (altricial birds) defecate inside the nest.
Nests of mute swans (Cygnus olor) were recorded on the edge of reed beds of the common reed (Phragmites australis) and rushes of the broadleaf cattail (Typha latifolia).The nests were stable, large, spherical, 95-161 cm in diameter, 60-140 cm high, at ca. 60 cm immersed in water, wet.The lining was indistinguishable from the rest of the nest due to rotting.The nests were built mostly from rotting parts of the common reed (Phragmites australis) and cattails Typha sp. as well as willow twigs (Salix sp).non-plant material consisted of high amounts of excrements of chicks and adult birds as well as chick eider as chicks spend much time in the nest (although the mute swan is a precocial bird), feathers of adult birds, shells and membranes of hatched eggs (birds do not remove eggshells from the nests).
Nests of coots (Fulica atra) similary to the nests of mute swans were recorded in reed beds of the common reed (Phragmites australis) and rushes of the broadleaf cattail (Typha latifolia) similarly to the nests of mute swans.They were not very big: Abbreviation: H -high; S -small; A -absent 24-31 cm, spherical, equipped with a type of "pier" providing easier access to water.They were mostly built from stems of broadleaf cattail (Typha latifolia) with an addition of sweet flag (Acorus calamus), fennel pondweed (Potamogeton pectinatus), sporadically containing twigs of black alder (Alnus glutinosa) and poplar (Populus sp.).The inside of the nests was lined with leaves of common reeds (Phragmites australis) and grasses (Poacae) poorly distinguishable from the rest of the nest.Very small amounts of feathers and faeces in comparison with those recorded in the nests of mute swans were observed.
Nests of great crested grebes (Podiceps cristatus) were recorded on the edge of a Phragmites australis reed bed growing on pond banks.They were unstable, floating, quite large, 40-68 cm in diameter, 10-19 cm high, mount-like in shape.Most of the nest structure was immersed in water; only a layer of 4-7 cm was above the water surface.The lining was hardly distinguishable from the rest of the nest.The nests were built from rotten plant material in which perennial dicots, a small amount of Canadian waterweed (Elodea canadensis), rushes (Typha latifolia) and fennel pondweed (Potamogeton pectinatus) were identified.More permanent plant material consisting of common reeds (Phragmites australis) formed the base of the nest.Animal material was rarely found in the nest structure: fish scales and bird feathers.Excrements were not found (a precocial bird).
Nests of black-headed gulls (Larus ridibundus) nesting in colonies and in setters were recorded in the centre of broadleaf cattail rushes (Typha latifolia).The nests were stable, mound-like, quite dry, 27-55 cm in diameter, 7-24 cm high.They were mostly built from remains of aquatic plants, usually broadleaf cattail Typha sp. and common reed (Phragmites australis).The base was made of twigs of willow Salix sp., black alder Alnus glutinosa, downy birch Betula pubescens, other birches (Betula sp.) and European black elderberry Sambucus nigra.Rhizomes of Agropyron sp., reed leaves and water horsetail (Equisetum fluviatile) were recorded in the lining which was not always well distinguished from the rest of the nest.Very small amounts of feathers and fish scales were found in the nest structure.
The nest of common gulls (Larus canus) nesting on a lake was recorded on a floating island built from rotten vegetation.The nest was mound-shaped, ca.30 cm in diameter, consisting of rotten, unidentifiable vegetation.It contained very few feathers and little excrements.
Nests of common terns (Sterna hirundo) were recorded on floating vegetation, mostly water lilies (Nymphaea sp. and Nuphar sp.).They were also mound-shaped, ca.20 cm in diameter.They consisted of rotten, unidentifiable vegetation.They contained very few feathers and slightly more excrements than the gull's nest.
The nest of black tern (Chlidonias niger) nesting in rushes of Typha sp.The nest was a small mound (ca.20 cm in diameter) made of broken stems of reeds and rushes.The nest did not have any excrements and had very few feathers.
Isolation and identification of fungi.Keratinophilic fungi were isolated with the keratin baiting method using white chicken feathers as the substrate.A total of 390 plates were made.Plates were filled with the nest material broken into smaller pieces to ½ and sterile feathers were placed on top.Feathers were sterilised using the method of ethylene oxide gassing as described in a study by Korniłłowicz (1994).Ten plates were prepared from each nest with the exception of nest 2 (Circus aeruginosus) from which 20 plates were prepared.nest material ranging from 200 to 500 g was selected randomly from ten different sites in the nest when the brood chamber was poorly defined or not evident or from its three layers (nest 2, Circus aeruginosus) comprising the lining, the outer layer and the layer in between (middle layer) (Pugh 1966).
Plates with the nest material were placed in a humidity chamber and incubated at 26°C for tree to four weeks.The forming mycelium layers were plated onto plates with Sabouraud glucose agar with actidion and chloramphenicol obtaining clean fungal cultures by passage.
The genus and the species of the fungi were identified using macroscopic characters on plates or microscopic characters in microcultures.Preparations of the mycelium developed on feathers were made in a drop of water to identify teleomorphs, which was particularly important for heterotallic species.The fungi were determined using systematic studies by: Ellis (1971); Domsch et al. (1980); van oorschot (1980); Currah (1985); Peberdy (1987).
Determination of physical and chemical properties of the nests.Water content in the nest material was determined with the weight method at 105°C.pH in H 2 O and KCl were measured potentiometrically.Total carbon and total sulfur content was determined with an elemental analysis by combustion analysis and in a thermal conductivity detector, C organic content by Thiurin method.The content of total n, total P, K, Ca, Mg was determined after sample mineralisation using the wet assay method in a mixture of concentrated H 2 sO 4 and perhydrol using flow spectrophotometry (n-tot., P-tot.) and with the atomic absorption spectroscopy method (K, Ca, Mg).
Results assessment.The number of plates (samples) with the nest material showing growth of keratinophilic and non-keratinophilic fungi (an arbitrary term) was used for the general assessment of the occurrence frequency of fungi.It was accepted that one plate can be colonised by only one strain of a fungal species.
The species diversity of fungi based on the number of isolates of fungi representing individual species was analysed by calculating Simpson's index (Krebs 1994) according to the formula: where p i is the share of isolates (strains) of species "i" in a fungal community and is the quotient of the number of strains of the species and the number of isolates of all fungi obtained on an isolation medium.Values of Simpson's index range from 0 to 1-1/S, where S is the number of species in a community of fungi.
The species dominance (Trojan 1975) was determined using the formula D = 100 .(Sa : S) where Sa -the sum of isolates of species a, S -the sum of isolates of the group.The group dominance (geophilic dermatophytes and Chrysosporium) was determined in a similar way, where Sa -the sum of isolates in a group, S -the sum of isolates of all fungi.
Correlation coefficients (r) were calculated to define the relationship between the frequency of dominant fungal species and some physical and chemical properties of the nests.

REsULTs
Physical and chemical properties of the nests.The analysis shows (Tab.3) that the level of humidity was very high and exceeded 80% in the majority of the nests (30 out of 38).Lower humidity was recorded only in the nests of grey heron and marsh harrier.A probably secondary increase of humidity was observed in the majority of the nests of grey heron (no 4-8) collected from the ground surface where they had fallen after a storm.The pH of the nests was close to neutral or slightly alkaline The level of total carbon and organic carbon recorded in the nest material varied (Tab.4).C organic content (in % of dry weight) ranged from 24.1% to 47.9%.Total n level was high or sometimes very high ranging from 1.28% d.w. to 5.38% d.w.A high content of total n was particularly high in the nests of grey heron: 2.29%-5.38%,which should be attributed to the accumulation of excrements from young birds.Different levels of phosphorus and calcium were recorded in the nests.A very high phosphorus content (7.2% d.w.) was observed only in some nests of grey heron.A high level of calcium, as high as 6.92% d.w., was recorded in some nets of mute swan, great crested grebes, black-headed gull and common tern.Total S content The data given in Tab. 5 shows that non-dermatophytic fungi representing the Chrysosporium group were the dominant group.A total of 273 strains were recorded, which corresponded to 78% of all keratinomycetes identified in the study.The remainder (22%) of the community of keratinophilic fungi was represented by geophilic dermatophytes (75 strains).
Generic and species diversity of keratinophilic fungi. of the nine genera isolated from the nests, four (Microsporum, Trichophyton and their teleomorphs Nannizzia and Arthroderma) represented geophilic dermatophytes and five represented non-dermatophytic fungi: Chrysosporium and Myceliophthora together with teleomorphs Arthroderma, Aphanoascus and Ctenomyces (Tabs 6-8, Fig. 2).The genus  The distribution of keratinophilic fungi and nest properties.Data on the occurrence of individual species of geophilic dermatophytes and Chrysosporium in each of the 38 nests are presented in Tables 7 and 8.They show a non-uniform distribution of the populations of keratinophilic fungi in the microhabitat: their occurrence was observed in some nests while they were absent in others.This corresponded to Grey heron   The most numerous species within both communities of keratinophilic fungi, Chrysosporium keratinophilum together with its teleomorph Aphanoascus fulvescens, showed preferences for habitats characterised by a very high humidity, which was recorded in the case of coot's nests, great crested grebe's nests as well as the nests of both species of gulls and terns (Tab.8).Apart from the above species, Chrysosporium tropicum, one of more frequent species, mostly colonised grey heron's nests while its occurred rarely or sporadically or did not occur at all in others (Tab.8).
It was shown that the frequency of T. terrestre (together with the teleomorph) is negatively correlated with the nest's humidity level and that of Ch. keratinophilum is positively correlated with it.An even stronger and positive correlation was observed between the frequency of occurrence of T. terrestre and the nest's pH and phosphorus content.The frequency of both species: Ch. keratinophilum (together with the teleomorph) and T. terrestre (together with the teleomorph), was also significantly positively correlated with the calcium content in the nests although the correlation coefficients were lower than those for pH and phosphorus content (Tab.10).
The colonisation rate of the nests and the species composition of so-called nonkeratinophilic fungi growing on feathers.The colonisation rate of the nest material by ubiquistic fungi (polyphages), arbitrarily called non-keratinophilic, varied greatly and ranged from 12% to 95%.The greatest number of ubiquistic fungi able  to colonise feathers occurred in the nests of grey herons and marsh harriers, and the smallest number was recorded in the nests of great crested grebes and mute swans (Fig. 1).Unlike keratinophilic fungi, the colonisation rate of native keratin by ubiquistic fungi corresponded to the richness and frequency of their species (Fig. 1, Tab. 11).The greatest richness and species diversity was observed in the case of non-keratinophilic fungi colonising the nests of grey herons and marsh harriers: 19 and 11 species and 50 and 39 strains, respectively (Tab.11).The smallest species differentiation of the biota of ubiquistic species colonising feathers was observed in the nests of both tern species: common tern and black tern, 5 and 3, respectively, represented by single strains (Tab.11).
As regards the species composition, ubiquistic fungi recorded in the nests and colonising native feathers were represented by 34 species belonging to 20 genera (the species of ten isolates was not determined).The most frequently isolated genera were Aspergillus, Gliocladium,Paeciliomyces,Penicillium and Scopulariopsis (Tab. 11).Similarly to keratinophilic species, individual species of non-keratinophilic fungi showed preferences for nests of specific bird species.Scopulariopsis brevicaulis was most frequently isolated from the nests of grey herons and marsh harriers: 15% and 20%, respectively, and Aspergillus fumigatus: an 18%-share within nonkeratinophilic fungi colonising these nests.Additionally, Doratomyces microsporus was frequently isolated from marsh harrier's nests on feathers (15% respectively).Among other ubiquistic species, two polyphagous species: Gliocladium catenulatum and Verticillium lecani, occurred as co-dominant species in black-headed gull's nests: the colonisation rate of feathers was 28% and 25%, respectively.on the other hand, Chaetomium globosum, which represented ca.63% of total non-keratinophilic fungi, was an accompanying species of feather colonisation by typically keratinophilic fungi in mute swan's nests (Tab.11).

DIsCUssION
The present study shows that keratinophilic fungi colonised 86.8% of the nests of wetland birds.A slightly higher (ca.5%) occurrence frequency of keratinomycetes was recorded only in nest boxes (Hubalek et al. 1973).The occurrence frequency of keratinophilic fungi, however, was higher in comparison with open-cup nests of land fungi.Hubalek et al. (1973) demonstrated the presence of keratinophilic fungi in 72.7% of such nests, mostly belonging to Passeriformes.The investigations also showed a high (76% on average) colonisation rate of the nest material by keratinophilic fungi.The nest material of marsh harriers (49%) was the least colonised and that of both tern species (90%) was the most strongly colonised material.
The widespread distribution of keratinophilic fungi in the nests of wetland birds was conditioned by the presence of the birds (breeding) and keratin matter, mostly feathers and less frequently hair, animal food remains, excrements and pellets.A considerable accumulation of total nitrogen as well as phosphorus and calcium indicated nest contamination with remains of animal origin.Both the nutrient factor (keratin) and high humidity as well as neutral to alkaline pH (pH 6.5-7.8) of the nest material were favourable for the development of keratinophilic fungi in the nests (Tab.2).As previous investigations show (Korniłłowicz-Kowalska 1997), keratinophilic fungi grow well on surfaces of feathers which are a non-wettable substrate when such substrate is in contact with water.The process is intensive when the substrate's pH ranges between 6.5 and 7.8 (Korniłłowicz-Kowalska, Bohacz 2002), which is connected with the optimum of extracellular keratinolytic proteases of these fungi (Korniłłowicz-Kowalska 1999).Similar observations were made by Kunert (2000) in relation to biodegradation of hair by keratinolytic fungi.
on the whole, a high richness of kertinomycete species was observed in the nests of wetland birds examined: altogether 22 species belonging to nine genera were recorded.A total of no more than 15 species of keratinomycetes is regularly isolated from natural environments such as the soil (Gueho, Villard and Guinet 1985;Korniłłowicz-Kowalska, Bohacz 2002).However, a high differentiation of the composition and the frequency of keratinomycete species colonising the nests was observed in the investigations depending on the species of the nesting bird.The greatest number of fungal species and their diversity, was observed in the nests of mute swans and coots and, further, of march harriers and grey herons.The smallest number of species and the lowest Simpson's indices were recorded in the nests of both tern species and black-headed gulls.Great differences in the species composition of keratinophilic fungi in nests depending on the bird's species were previously demonstrated by Hubalek (1974) in his analysis of terrestrial birds, mostly Passeriformes.
It is interesting that the nests in which the greatest richness and diversity of keratinomycete species were observed (mute swans and coots) differed considerably by keratin matter content (feathers).High amounts of feathers and excrements were observed in mute swan's nests while small amounts were noted in the coot's nests or they were absent (Tab.2). on the other hand, the two birds' species had similar breeding biotopes and feeding grounds.Both colonized fertile reservoirs (ponds), built nests in reed beds and broadleaf cattail rushes, and mostly fed in the littoral zone (mute swans also in the middle of the ponds), feeding on vegetation and small invertebrates (snails, insects) occurring on plants and the bottom slime of shallow waters.A high rate of contamination of the nests by geophilic keratinophilic fungi may also have been connected with the contamination of the feeding grounds of these birds by the fungi.A high accumulation of geophilic keratinophilic fungi is observed in bottom sediments and reservoir waters affected by strong anthropopressure such as ponds (Korniłłowicz 1993;Ulfig 1986Ulfig , 1987;;Ulfig, Ulfig 1990; Ulfig et al. 1996).Allochthonic organic substances, including keratin remains (feathers, hair, etc.), are a source of these fungi (Korniłłowicz 1993;Ulfig et al. 1996).It is highly probable that the fungi may have been mechanically transferred on the plumage or collected by the birds with the food.The fact that the frequency and the distribution of keratinophilic fungi on the surface of birds' bodies depends on their feeding habitats was previously reported by Pugh (1965Pugh ( , 1966)).
Bird excrements may also have been a source of keratinophilic fungi in the nests of many bird species.Faeces contamination was observed especially in the case of grey herons, mute swans and marsh harriers (Tab.2).The occurrence of keratinophilic fungi in excrements and their spread by excretion with faeces have been reported by, e.g., Dominik, Majchrowicz (1970);nooruddin, Singh (1987); Garetta et al. (1992).
The thesis that feeding habitats and "animalisation" (enrichment in keratin remains) are mostly a source of keratinophilic fungi in the nests of wetland birds is also corroborated by the observations of breeding biotopes and feeding grounds of other bird species examined in the study.Few species of keratinophilic fungi were recorded in the nests of these birds (great crested grebe, black-headed gull, common tern and black tern).Bird feathers or excrements were also observed in them sporadically.Apart from black terns, these birds are piscivorous, search for fish in the water, diving into it (great crested grebe) or catching fish from the air (blackheaded gull, common tern).Black terns, on the other hand, are insectivores and catch insects in flight, over the water surface and fields.This manner of feeding and the types of feeding sites (in the water, air above the reservoir) are not favourable for the acquisition by birds of geophilic keratinophilic fungi that can occur in these environments only accidentally.
The majority of bird species examined did not come into contact with the soil (an environment believed to be a major reservoir of geophilic keratinophilic fungi) or such contact was rare.Marsh harriers which often hunt outside the breeding site, in meadows and fields, catching small mammals, lagomorphs, sometimes poultry, and grey herons which supplement their diet with voles outside the breeding season, were the only exceptions.Little importance of the soil as a source of contamination of the plumage and nests of the majority of fungi was previously reported by Pugh (1966) and Rees (1967).
our examinations show that non-dermatophytic keratinophilic fungi of the Chrysosporium group are a dominant group in the nests of wetland birds.They represented ca.78% of the keratinophilic mycobiota of the nests (273 nests), while the genus Chrysosporium itself constituted ca.63%.The dominance of Chrysosporium in the nests of land birds was reported by Hubalek (1974) andHubalek et al. (1973) several times.In their analysis of the nests of passerines, mostly Eurasian tree sparrow Passer montanus, Hubalek et al. (1973) showed that "chrysosporia" constituted over 90% of keratinomycete populations in the nests.Dermatophytes ranged only from ca. 2% to ca. 9% of the fungi (Hubalek et al. 1973).The share of geophilic dermatophytes was between 0% (both species of terns and gulls) and 44.3% (mute swan) in the nests of wetland birds studied.
A generally higher frequency of Chrysosporium in comparison with geophilic dermatophytes in birds' nests may be connected with their higher occurrence in the plumage and on birds' feathers (Hubalek 2000) and a lower keratinolytic activity (Korniłłowicz-Kowalska 1997;Kunert 2000).Due to the latter, these fungi grow better in environments containing more accessible keratin sources, such as feather keratin rather than, for instance, hair keratin.Moreover, nest pH (pH in H 2 o 6.5-7.8) was a factor favourable for a high frequency of Chrysosporium in the nests of wetland birds examined.The majority of Chrysosporium species prefer environments with a higher pH and are alkalotolerant (Kushwaha 2000).
Ch. keratinophilum was the most frequently isolated species from the nests of wetland birds.Together with its telefomorph (Aphanoascus fulvescens), it colonised the nests of all the birds and its share in the community of keratinophilic fungi was 53% on average.Ch. tropicum (11.5% of total keratinomycetes), isolated mostly from the nests of grey herons, was less widespread.According to Hubalek (1974), A. fulvescens mostly colonises nests of wetland birds and Ch.tropicum is a frequent coloniser of these nests.
Trichophyton terrestre, which together with its teleomorphs (Arthroderma quadrifidum and A. insingulare) constituted 12.5% of all isolated fungi, and Microsporum gypseum and its telefomorph (Nannizzia gypsea), which constituted 7.1%, had the highest frequency among geophilic dermatophytes.Populations of T. terrestre mostly colonised the nests of marsh harriers and grey herons while M. gypseum colonised the nests of mute swans.
The frequency and distribution of individual keratinomycete populations in the nests of wetland birds was conditioned primarily by the differences in the humidity and pH level of the nests.A similar phenomenon was observed in a study on the frequency of keratinomycetes in the soil by Chmel et al. (1972) as well as by Korniłłowicz (1993) and Korniłłowicz-Kowalska, Bohacz (2002).Soil pH was the most important selection factor in the populations of these fungi (Korniłłowicz 1993;Korniłłowicz-Kowalska, Bohacz 2002).
The present investigations show that a high humidity of the nest material was the reason for the accumulation of Ch. keratinophilum in the nests of wetland birds.The occurrence frequency of the fungus increased as water content increased (r=0.62,p=0.05).The colonisation of nests with a high humidity level (ok.62%) by Ch. keratinophilum was also observed by Hubalek et al. (1973), who reported that Ch. keratinophilum (as a teleomorph) is isolated more frequently from the plumage of water birds than land birds.Ch. keratinophilum's preference for environments with a high level of humidity results from its hygrophilous (hydrotolerant) nature, which is related to a high demand for water (Garg et al. 1985;Hubalek 2000).Ch. keratinophilum is also an alkalotolerant species.A reverse relationship with the humidity level in the nests was observed in the population of Trichophyton terrestre, a species belonging to xerophyles (Garg et al. 1985).The frequency of occurrence of this dermatophyte decreased together with an increase in the water content in the nest material (r=-0.61,p=0.05).T. terrestre's preference for dry environments was also observed by Chmel et al. (1972) and Chmel & Vláčiliková (1975).
The present investigations also confirm growth stimulation of T. terrestre in alkaline environments previously observed by other authors (Chmel et al. 1972;Ulfig et al. 1996).The frequency of this dermatophyte increased as pH increased (r=0.81,p=0.05) reaching its maximum in the nests of grey herons in which pH in H 2 O was ca.7.4-7.8.In the case of T. terrestre, a high content of n-total, phosphorus and calcium was also a factor significantly conditioning its frequency of occurrence in the nests.It may be supposed that a high level of these elements contributed primarily to a pH increase in the nests.nest alkalisation was caused by the release of ammonia produced during the ammonification of uric acid contained in bird faeces (accumulated in very high amounts in the nests of grey herons) and calcium ions and phosphates from the digestion of animal food (fish) and excreted in faeces.The relationship between the frequency of T. terrestre and the calcium level in the environment (soil) was reported by Chmel et al. (1972).
Microsporum gypseum is also interesting in the group of other, more frequently isolated species of keratinophilic fungi.Although no significant correlations between its frequency of occurrence (the number of samples with fungal growth was too small) and the physico-chemical properties of the nests were observed, its occurrence limited mostly to the nests of mute swans and coots may suggest preferences for environments with neutral pH and a relationship with biotopes polluted with organic matter.M. gypseum is a dominant dermatophyte species in bottom sediments of waters strongly polluted by communal waste waters delivering considerable amounts of keratin matter (Ulfig 2000).Similar observations were made on the occurrence of M. gypseum in soils polluted with waste waters (Ali-Stayech, Jamous 2000).Previous analyses (Korniłłowicz-Kowalska, Bohacz 2002) of the occurrence and the distribution of geophilic dermatophytes and Chrysosporium in soils with different physico-chemical properties showed that M. gypseum (as well as M. cookei) colonises exclusively soils characterised by a considerable "animalisation" and neutral pH.
It should be stressed that the majority of keratinomycete species recorded more frequently in the nests of wetland birds were thermotolerant fungi such as Ch.keratinophilum, Ch. tropicum, M. gypseum.They grow well at a temperature of 37°C and maximum growth temperatures are 40-41°C (Garg et al. 1985).This is consistent with nest temperatures during incubation reaching a maximum of 40-41°C (Pinowski et al. 1999).
on the other hand, species considered to be typical soil species such as Trichophyton ajelloi and Ctenomyces serratus (Domsch et al. 1980) were rare in the nests.neutral or alkaline pH of the nests of wetland birds did not encourage the occurrence of acidophilic species such as T. ajelloi, Arthroderma uncinatum, A. curreyi (Garg et al. 1985).The exception was Chrysosporium tropicum, which is thought to be acidophilic according to Garg et al. (1985).It occurred relatively frequently in the nests of grey herons where pH (H 2 o) was from 5.99 to 7.76.Hubalek et al. (1973) also reported the occurrence of Ch. tropicum in nests with pH ranging from acidic of slightly alkaline (pH 5.5-7.5).It is possible that the species colonises biotopes with a broad pH range, adapted mostly to high temperatures (van oorschot 1980) and resistant to light.Extreme conditions are observed in grey heron's nests during breeding: low humidity, sun exposure and additional nest heating related to it.This allows only species of keratinomycetes, such as T. terrestre and Ch.tropicum, that are most resistant to the lack of water and insolation, to survive. of the species of keratinophilic fungi recorded in the nests of wetland birds, a widespread occurrence and a high frequency of Ch. keratinophilum in many nests and the accumulation of M. gypseum in the nests of mute swans may raise greatest concerns.Ch. keratinophilum and its teleomorph Aphanoascus fulvescens are opportunistic non-dermatophitic causal agents of mycoses in humans and animals (Gueho et al. 1985).Microsporum gypseum is the most virulent geophilic dermatophyte causing inflammatory tinea corporis and tinea capitis in humans (Hayashi, Toshitani 1983;offidani et al. 1998).It also causes mycoses in animals (Garetta et al. 1992).
The results obtained in this study also confirmed the occurrence of populations of potentially pathogenic ubiquistic moulds, Aspergillus fumigatus and Scopulariopsis brevicaulis, in the nests, observed in a previous study (Korniłłowicz-Kowalska, Kitowski 2009).The species are thermotolerant and alkalotolerant, and show keratinolytic abilities (Kozakiewicz, Smith 1994;Santos et al. 1996;Filipello-Marchisio et al. 2000).Pathogenic strains of these fungi cause lung aspergillosis (A.fumigatus) and onychomycosis (S. brevicaulis) (Dvořak, otčenašek 1969).Aspergillus fumigatus is very frequently isolated from nests of birds, their ontocoenoses, plumage and pellets (Hubalek 1974;Kruszewicz et al. 1995;Shin et al. 1996;Korniłłowicz-Kowalska, Kitowski 2009).It is the most frequent causal agents of mycoses, mostly of the lungs and air sacs in wetland birds (Mikaelian et al. 1997).The present investigations show that nests are a potential source of pathogenic infections with A. fumigatus in wetland birds.They are also some of reservoirs of geophilic fungi causing dermatomycoses and systemic mycoses in humans and mammals.These fungi can penetrate the water body from the nest and can be transferred by birds over considerable distances during migration. 37

Table 1
Bird species, nest location and collection date

Table 2
Animal matter ratio in the nest structure (in relation to the nest mass)

Table 3
Humidity level (in % of dry weight) and pH level in the nests pH in H 2 o 6.55-7.76)with the exception of one nest of grey heron (no 7) where a weakly acidic pH was recorded (pH in H 2 o 5.99) (Tab.3).
Abbreviations: 1 -the outer layer of the nest; 2 -the intermediate layer of the nest; 3 -the inner layer (lining) of the nest (

Table 4
The content of some macroelements (in % of dry weight) in the nest material

Table 5
numbers of isolated genera, species and isolated of geophilic dermatophytes (GD) and the Chrysosporium group (Ch)

Table 6
A list of geophilic dermatophytes and Chrysosporium isolated from the nests of wetland birds Abbreviations: (+) yes; (-) no

Table 7
The frequency and distribution of individual species of geophilic dermatophytes in the nests of wetland birds

Table 9
Simpson's index of species diversity (D) for communities of keratinophilic fungi in the nests