Annals of RSCB Vol. XVII, Issue 1/2012

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1 COMPARATIVE STUDIES OF MORPHOLOGY AND ULTRASTRUCTURE IN TWO COMMON SPECIES OF DERMATOPHYTES: MICROSPORUM CANIS AND MICROSPORUM GYPSEUM C. V. Mihali 1, A. Buruiana 2, Violeta Turcus 1,2, Aurelia Covaci 1, A. Ardelean 1,2 1 INSTITUTE OF LIFE SCIENCES, UVVG ARAD 2 THE FACULTY OF NATURAL SCIENCES, ENGINEERING AND INFORMATICS, UVVG ARAD Summary The paper presents the ultrastructural features of the macroconidia, microconidia, talus and general aspect of mycelium in two common species of dermatophytes, Microsporum canis and Microsporum gypseum.these species belongs to Arthrodermataceae family, order Onygenales of Ascomycota division from Fungi. Observations were carried out on fragments of fungal dermatophytes culture media containing strains isolated from patients with clinical suspicious of tinea corporis and tinea capitis. The microscopic examination of colonies and macro/microconidia aspects are essential in species diagnosis. In Microsporum canis and Microsporum gypseum species, the morphological aspects can be analysed very precise using ESEM (environmental scanning electron microscopy) investigations and light microscopy. Keywords: dermatophytes, ESEM, ultrastructure, morphology, Microsporum. mihaliciprian@yahoo.com Introduction Dermatophytes are a group of molds that are related morphologically and physiologically between them, causing infections in humans and animals: tinea capitis and tinea corporis (De Vroey, 1895). They possess two important properties: they are keratinophilic and keratinolytic. This means that their have the ability to digest keratin in vitro in saprophytic state and to use as a substrate and some of them can invade tissues in vivo and cause tineas. The morphology of dermatophytes in culture, for nomenclature purpose can be divided in two states (based on stage in life cycle): anamorphic state and teleomorphic states. The anamorphic state is the asexual state where somatic reproduction is present and has a particular morphology used in taxonomy description for these dermatophytes. The teleomorph represent the sexual state, differentiated from the anamorph (Ajello, 1977). The dermatophytes have been divided (Georg, ) into three ecological groups: geophiles, zoophiles and anthropophiles. The two species of dermatophytes, Microsporum canis and Microsporum gypseum, belongs to Arthrodermataceae family, order Onygenales of Ascomycota division from Fungi The genus Microsporum produces microconidia and macroconidia. Macroconidia presents 3-5 septa with a thin or thick echinulate cell wall, spindle shaped or rounded ends (Vismer et al., 1987). The important distinguishing structure of this genus is the echinulations of the macroconidial cell wall. The thickness of the macroconidia and shape varies depending on the species. Microconidia are pyriform or clave and has 2-3µm lenght. M. gypeseum is geophillic and has been found in soil all over the world (Akin and Michaels, 1972). The telomorph (the sexual form of a fungus is Arthroderma gypseum ( Weitzman, 1995). Microsporum canis is the main agent

2 of dermatophytosis in dogs and cats, frequent responsable for zoonosis. This organism can cause tinea capitis-corporis ( Walter and Dorinda, 1987) in humans, simple ringworm and can be asymptomatic in pets (Shoichi et al., 1986). The major reservoir Material and methods Were examinated 4 strains of Microsporum canis and Microsporum gypseum from patients with isolated cutaneous lesions of tinea corporis and tinea capitis. The fragments of fungal dermatophytes culture media containing strains isolated from patients with clinical suspicious of tinea corporis and tinea capitis, were examinated using optical microscopy and (ESEM) environmental scanning electron microscopy (Ludwig, 1998; Patrick, 2009), for morphological and structural particularities studies (Glauert, 1974). Light microscopy 1. Native samples was made using adhesive-tape method of sampling, the morphology studies was made using a BX 43 Olympus optical microscope with semi plan apochromate objectives 10X, 20X, 40X, images were captured using a digital CCD camera, CX3 Olympus. 2. The stain method used was with one drop of lactophenol cotton blue between the glas Results and discussions In both species of dermatophytes examinated, they showed an abundance of fructification (Fig.1- E, F, G, H) characterised by many specific macroconidia. In native samples, in situ we saw poor details and these cannot be used for identification. The macroconidia are well represented but the structural details, (for instance echinulations) are difficult to observe. In Microsporum canis after a few days the colony aspect is more fluffy and has a central depressed area, and showed radial folds. The pigment undersurface of the colony was yelloworange (Fig.1- A, B). In Microsporum gypseum, we observed a large number of 86 in companion animals is the domestic cat and dog (De George et al., 2001). The species teleomorphic states is represented by Arthroderma otae, Hasegawaz and Usui, slide and adhesive-tape. ESEM with cooling stage Colony sample of Microsporum gypseum and Microsporum canis were excised and placed in Eppendorf tubes with Glutaraldehyde 2,7% for 6 hr at 4ºC. After they were mounted into the Cooling stage (which is a temperature controlled specimen holder of ESEM microscope) which has 3 C and 100% RH. The morphology studies was made using Fei Quanta 250 ESEM, the parameters of ESEM examination were represented by the following items: the image mode, the working distance (WD), the beam conditions, chamber pressure and the relative humidity into sample. In the same order they were: the gaseous secondary electron detector detector (GSED); the working distance: 5 mm; the beam conditions: 12,5 15 kv, spot 4,5; the pressure range: 910 to 1400 Pa and the relative humidity of 100%. macroconidia /microconidia and a long talus with many regions with microconidia (Fig.1 G, H). The hilar appendix from macroconidia was not observed. Microconidia aspect was clavate, macroconidia with 3-6 septumuri prominently present in native preparations. The cultural aspect revealed the presence of cotton-like white colonies, with yellowish brown central area and radial ridges, with the reverse of the colonies being yellowish. In ESEM examinations in both species, microconia/macroconidia were well represented (Fig.2). Microsporum gypseum presented talus with microconidia, arthrospors and macroconidia.

3 Microconidia are clavate in shape, small, unicellular, thin wall without showing echinulations on their surface. Macroconidia number is significantly higher. The talus ramifications were well represented as direct growth of tall. Macroconidia in Microsporum gypseum were ellipsoidal shape / pear. It presents 3-6 septa, echinulations distribution (spins) are relatively uniform distributed across its surface. Echinulations seemed to be easy sunked into macrooconidia surface. Echinulations are present on the talus surface also (Fig.2- E,F). Were observed the presence of arthrospors with specific barrel form and its upper part presents a birth scar. The species Microsporum canis mycelium is well developed, has many cordage, macroconidia presence were lower that in Microsporum gypseum. Macroconidia spindle-shaped form with echinulations arranged more uniformly, predominant in their extremities. Distal extremity is sharp and ends with a button. Septa number are well represented compared with Microsporum gypseum and they appear larger. Echinulations were well defined and not submerged as in Microsporum gypseum macroconidia occur. Microconidia development is presented in a single direction from the talus, they appear more elongated as they were in Microsporum gypseum. Fig. 1 - Development and cultural aspect in Microsporum canis (A-obverse B reverse view) and Microsporum gypseum (C- general aspect, D-colony morphology); E Microsporum canis general aspect (ob.10x), F - macro/microconidia (ob.100x); G Microsporum gypseum macroconidia (ob.100x), H - macro/micro-conidia, talus (ob.100x). 87

4 Fig.2 General aspect of Microsporum canis (A) /Microsporum gypseum (B) mycelium. Macroconidia in Microsporum canis(c) and Microsporum gypseum(e). G-H talus aspect / microconidia in Microsporum canis. I-J microconidia and talus/macroconidia with numerous echinulations in Microsporum gypseum. D - explanatory scheme for difference between macroconidia in the two species. F - explanatory scheme for image E. 88

5 Conclusions By using light microscopy examination, morphology could be achieved at the two dermatophytes species without the possibility of studying the details of ultrastructure. In ESEM analysis, macroconidia from both species were investigated with emphasizing the differences between them. Echinulations were easily submerged on surface of macroconidia in Microsporum gypseum species and present well marked on surface of macroconidia in Microsporum canis species. Another morphological aspect to note is represented by the presence of echinulations on talus surface at Microsporum gypseum species. Acknowledgements This work was cofinanced from the European Social Fund through Sectoral Operational Programme Human Resources Development , project number POSDRU/89/1.5/S/63258 Postdoctoral school for zootechnical biodiversity and food biotehnology based on the ecoeconomy and the bio-economy required by eco-san-genesys. References Ajello L. Milestones in the history of medical mycology: the dermatophytes. In: Iwata K (Ed.) Recent Advances in Medical and Veterinary Mycology, Proceedings of the sixth Congress of the International Society for Human and Animal Mycology, University of Tokyo, Tokyo, Akin D.E., Michaels G.E., Microsporum gypseum macroconidial development revealed by transmission and scanning electron microscopy, Medical Mycology, Vol. 10, No. 1, Pages 52-55,1972. De George H. M.,Robert W. K.,Danny W. S.,Craig E. G.,Small animal dermatology,elsevier Health Sciences, ISBN , 1465 pp., De Vroey C. Epidemiology of ringworm (dermatophytosis). Seminars in Dermatol.; 4: , Georg L.K. Animals in public health: Diagnosis and Nature. US Department of Health, Education and Welfare. Public V 89 Health Service, Bureau of State Services Communicable Disease Centre. Antlanta, Georgia Glauert M., Practical Methods in Electron Microscopy, vol. 3, North Holland, Amsterdam, pp l-l 10, Ludwig R., Scanning Electron Microscopy: Physics of Image Formation and Microanalysis, pp.449, ISBN , Patrick E., Handbook of Sample Preparation for Scanning Electron Microscopy and X- Ray Microanalysis,, pp. 323, ISBN , Shoichi S., Toshio K., Ennji T., Differentiation in the Frond and Boring Hypha of the Dermatophyte Microsporum canis Invading Human Hair in vitro, Electron Microsc. (Tokyo),35 (1), 8-46, Vismer H. F., Findlay G. H., Eicker A., The septal ontogeny, germination and electron microscopy of Microsporum gypseum macroaleurioconidia, Mycopathologia, Jun; 98(3): , Walter B. S., Dorinda E. S., The infected hairs of tinea capitis due to Microsporum canis: Demonstration of uniqueness of the hair cuticle by scanning electron microscopy, Journal of the American Academy of Dermatology, Volume 16, Issue 2, Part 1, February Pages ,1987. Weitzman, I., S. A. Rosenthal, and M. Silva- Hutner., Superficial and cutaneous infections caused by molds: dermatophytoses, p In B. B. Wentworth (ed.), Diagnostic procedures for mycotic and parasitic infections. American Public Health Association, Washington, D.C