Introduction

Keratinophilic fungi are an ecologically significant group of fungi that decompose keratin—animal-origin protein—which is one of the most abundant and highly stable proteins on earth. They utilize it as a nutrient substrate for growth. The distribution of these fungi depends on different factors, one of which, of vital importance, is human and/or animal presence [1]. Vanbreuseghem reported for the first time the presence of dermatophytes in soil [2] using the hair bait technique. Since then, researchers across the globe are using hair bait technique for the isolation of keratinophilic fungi from soil [3–8]. Some of these fungi are well-known dermatophytes and are known to cause superficial cutaneous infections (dermatophytoses) of keratinized tissues (skin, hair and nails) of humans and animals.

Kaziranga National Park (latitudes 26°30′N and 26°45′N and longitudes 93°08′E to 93°36′E) is one of the India’s finest parks and is situated in Assam, home to several endangered species. Its tropical climate and geographic diversity make it a potentially interesting area to study the distribution of keratinophilic fungi. We, therefore, undertook this study and reported the results obtained.

Materials and Methods

Collection and Processing of Soil Samples

Seventy-eight soil samples were collected from the various localities in the vicinity of Kaziranga National Park (Assam), India, during April to October 2009 (Table 1). The samples were taken from the elevated regions known as chapories, which provide retreats and shelter for animals during floods, soil from water coarse bank, forest soil, grassland soil, barren land soil, the raw road (pagdandi) soil and dropped off feathers. Soil samples were collected by removing a layer of soil not exceeding 3–5 cm in depth with a sterile stainless steel spatula and transferred in sterile polyethylene bags, brought to the laboratory and stored at 15 °C if not processed promptly. Keratinophilic fungi were isolated by the baiting technique of Vanbreuseghem [2] using human hair as keratin bait. For this, sterile Petri dishes, half filled with the soil samples and moistened with sterile tap water, were baited by burying sterile human hairs in the soil. These dishes were incubated at room temperature (25 ± 1 °C) and examined daily from the fifth day for fungal growth over a period of 4 weeks.

Table 1 Distribution of fungi in soil samples of Kaziranga National Park
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Isolation and Identification of Keratinophilic Fungi

The growth on hairs was observed under a stereoscopic binocular microscope and was subcultured on SDA (Sabouraud’s dextrose agar) plates amended with chloramphenicol (50 mg/l) and cycloheximide (500 mg/l). The plates were incubated for 5 to 10 days at room temperature. The isolated cultures were checked for purity and subcultured to obtain pure cultures. These fungi were identified based on the various available monographs (Sigler and Carmichael [9] Oorschot [10] Currah [11] Arx [12] Cano and Guarro [13]).

Molecular Identification of Keratinophilic Fungi

The DNA sequences of ITS1-5.8S-ITS2 region were used for molecular characterization of the cultures. Fungal genomic DNA was extracted [14]. ITS1 and ITS4 were used as the forward and reverse primers for amplification of ITS1-5.8S-ITS2 rDNA [15]. PCR amplification was performed in 100 µl reaction volumes containing 10X buffer 10 µl, MgCl2 (25 mM) 2 µl, dNTP (10 mM) 2 µl, ITS1 primer (20 pm) 2 µl, ITS4 primer (20 pm) 2 µl, Taq Polymerase (2.5U) 1 µl, DNA Sample (5 µg/ml) 3 µl and Milli Q water 78 µl. The PCR was carried out using a Thermal Cycler (M.J. research, PTC 200) with following conditions: denaturation for 5 min at 94 °C, 34 cycles of (30 s at 94 °C, 30 s at 55 °C, 1 min at 72 °C) extension for 4 min at 72 °C and storage at 4 °C. Controls were used in each set of reactions. The final products were analyzed by agarose gel electrophoresis (Sigma). The PCR products were sequenced using ITS1 and ITS4 primers at Geneombio Technologies Pvt Ltd, Pune, India, using Applied Biosystems 3730 DNA analyzer.

Phylogenetic Analysis

Similarity analysis of the nucleotides was performed by BLAST searches against sequences available in GenBank [16]. For phylogenetic tree construction, multiple sequences were obtained from GenBank and the alignments were performed using MEGA6 [17].

Results and Discussion

Data on the distribution of keratinophilic fungi in the soils of Kaziranga National Park (Assam) are given in Table 1. The data show that only 39 of the 78 samples yielded keratinophilic fungi. A total of eleven species from seven genera were isolated: Aphanoascus durus (1.28%), Arthroderma tuberculatum (3.84%), Arthroderma corniculatum (1.28%), Chrysosporium indicum (16.66%), C. tropicum (3.84%), Ctenomyces serratus (5.12%), Keratinophyton punsolae (1.28%), Microsporum appendiculatum (1.28%), Microsporum gypseum complex (11.53%), Trichophyton mentagrophytes (1.28%) and Trichophyton terrestre (2.56%).

All the eleven strains yielded unique PCR amplicon. The amplicon size of the ITS1-5.8S-ITS2 rDNA regions for the eleven strains was ranging from 409 to 571 bp, where C. indicum is smallest and T. terrestre is largest in size. The other species showed an amplicon size of approximately 600 bp. The sequences were compared with sequences deposited in the NCBI database for taxonomic identification, and a phylogenetic tree was constructed with the closely related type strain sequences based on rRNA gene sequences (ITS region) using the maximum composite likelihood method. The phylogenetic tree indicates different group of clusters for each strain as depicted in (Fig. 1). The phylogenetic tree indicates different clusters for each isolate showing their sequence variation. The sequences obtained show greater than 96% similarity to the reference sequences available in NCBI GenBank database. Two reference sequences from genus Aspergillus, viz. Aspergillus fumigatus and Aspergillus niger, were used to draw outgroup to the existing clades generated by all study strains. Outgroup forms distinct clade distantly placed outside the clades formed by eleven strains.

Fig. 1
figure 1

Phylogenetic tree based on ITS1-5.8S-ITS4 region sequences for various isolated strains with reference strains. Numbers at the respective nodes are percentage of 1000 bootstrap replicates. Bar indicates genetic distance due to sequence variation. The tree was rooted Aspergillus niger and Aspergillus fumigatus as outgroup

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Chrysosporium indicum (16.66%), the most dominant keratinophilic fungi in Indian subcontinent, was the most frequently isolated species. It is well adapted to warmer conditions of India [18]. Microsporum gypseum complex was the second most dominant fungi (11.53%). It has been reported from various parts of India [18–20]. Chrysosporium tropicum was 3.84% in distribution. It is a cosmopolitan species and reported from various parts of India [21–23]. Ctenomyces serratus was recorded from 5.12% samples. It has been reported from various parts of India [24, 25].

The other fungi recorded were Aphanoascus durus (1.28%), Keratinophyton punsolae (1.28%), Arthroderma tuberculatum (3.84%), Arthroderma corniculatum (1.28%), Microsporum appendiculatum (1.28%), Trichophyton mentagrophytes (1.28%) and Trichophyton terrestre (2.56%).

Microsporum appendiculatum is reported for the first time from India. Arthroderma corniculatum was reported for the first time from the soils of Conakry as Microsporon boulardii [26]. Later on, its perfect state was reported as Arthroderma corniculatum [27]. It is also reported from Sanjay Gandhi National Park, Mumbai [7].

Trichophyton mentagrophytes is well-known dermatophyte and known to cause superficial infections to man and animals. Trichophyton mentagrophytes has been reported from soils at public places at Gulbarga [18], primary schools and public parks of Madras [28], public park of Mumbai [29]. Another species of Trichophyton, i.e., T. terrestre, was reported from soils of Agra [30], glacier bank soils [31] and salt pan soils of Mumbai [3].

Arthroderma tuberculatum was recovered from soils of caves around Mumbai [32], birds of Orissa [33] and also from pigeon feather from the buildings of Thane [34]. Aphanoascus durus was recorded from Indian soil of Gir Forest National Park and Wildlife Sanctuary, Gujarat [35], Sanjay Gandhi National Park, Mumbai [7] and soils of Lonar crater [24]. Keratinophyton punsolae was reported from the soil of Spain [13] and from the soil of Bahrain [4]. We are reporting for the first time the presence of keratinophilic fungi from Kaziranga National Park Assam (India).

Keratinophilic fungi play a vital role in nature in the breaking down and mineralization of keratinous substrate into simpler substances. Their ability to grow on keratin regarded them as pathogens to human and livestock. For example, an invasive infection was noted in an 18-year-old woman who was a bone marrow transplant recipient as Chrysosporium, where the infection began as a facial swelling and extended into the central nervous system [36]. Chrysosporium tropicum was reported from comb lesion in two different breeds of chicken in India [37]. Chrysosporium zonatum was reported causing disseminated infection in a patient with chronic granulomatous disease [38]. In Japan, C. zonatum strains were also isolated from bronchial lavage from a female in Chiba and from a male in Kyushu. Both patients were with pulmonary cavities [39]. Recently, Chandrakar et al. [40] reported a case of subcutaneous fungal abscess over the great toe caused by a keratinophilic fungus, an unknown Chrysosporium sp., in a 60-year-old diabetic female. Thus, these fungi may be regarded as opportunistic pathogens.

Different Chrysosporium species have been identified in multiple reptile species that were presented with clinical signs of infection. Chrysosporium guarroi was described in pet green iguanas (Iguana iguana) from different geographic areas of Spain [41] and Turkey [42]. Chrysosporium queenslandicum has been reported in a garter snake [43]. Chrysosporium keratinophilum, C. tropicum and Chrysosporium spp. have been regarded as agents of cutaneous and systemic mycoses in reptile species, as reviewed by [44]. Nannizziopsis vriesii (CANV) has been identified as the cause of dermatitis in multiple reptile species such as chameleons [45], snakes [46, 47], salt-water crocodiles [48], bearded dragons [49, 50], green iguanas (Iguana iguana) [51] and girdled lizards [52]. Nannizziopsis vriesii was isolated from skin lesions in a pet bearded dragon [53], and Chrysosporium ophiodiicola was isolated from mycotic granuloma of a black rat snake [54]. Aphanoascella galapagosensis was recovered from carapace keratitis in a Galapagos tortoise residing in a south Texas zoological collection [55]. Most of the infections caused by these fungi have been described in the last 10 years and is unclear whether this could be attributed to recent climatic changes that could have affected the environment, where these animals live or that previous infections had been overlooked or misidentified [56]. Further studies and reports are needed to determine the importance of Chrysosporium spp. in captive and wild animals as well as the extent of its zoonotic capabilities.