8 l KEYWORDS Cervus elaphus, Dermatophytes, Fulgal flora, Prevalence, Red deer, Stelvio National Park PAGES 8 – 14 REFERENCES Vol. 1 No. 2 (2014) ARTICLE HISTORY Submitted: September 28, 2014 Revised: September 30, 2014 Accepted: October 03, 2014 Published: October 21, 2014 CORRESPONDING AUTHOR Roberta Perego, Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare (VESPA) - Università degli Studi di Milano Via G. Celoria 10, 20133, Milano, Italy e-mail: roberta.per ego@unimi.it phone: +39 02 50318188 fax: +39 02 50318171 JOURNAL HOME PAGE riviste.unimi.it/index.php/haf Prevalence of Dermatophytes in Red Deer (Cervus elaphus) in The Stelvio National Park, Italy. Roberta Pereg o1, Daniela Prov erbio1, Eva Spada1, Giada Bagnagatti De Giorgi1, Emanuela Valena1 and Elisabetta F erro1 1 Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare (VESPA), Università degli Studi di Milano, Milano, Italy. ABSTRACT. Dermatophytosis has been described in wildlife, but the literature reporting dermatophyte prevalence in deer is incomplete. To determine the prevalence of dermatophytes and to evaluate the hypothetical role of asymptomatic carriers hair samples were collected from 30 legally hunted wild red deer (Cervus elaphus) in the Stelvio National Park, Italy. All deer were visually examined for dermatologic lesions and the coat was brushed using a modified Mackenzie collection method. A small sample of hair was used for direct microscopical examination and subsequently fungal culture was performed on the hair samples. Macroscopic and microscopic examinations were used to identify dermatophytes, saprophytic fungi and yeasts. None of the deer had visible cutaneous lesions. No dermatophyte spores or hyphae were found on direct microscopical examination and, when hair samples were cultured, dermatophytes were not demonstrated in any sample. Only saprophytic fungi were grown, predominantly Alternaria spp., Mucor spp., Cladosporium spp. These results did not reveal the presence of asymptomatic carriers of dermatophytes in the deer sample population of Stelvio National Park and suggest that it is unlikely that, at least in the investigated geographical area, the red deer act a reservoir for transmission of dermatophytes to other wild animals, livestock or people living locally. R. Perego et al. - Int. J. of Health, Animal science a nd Food safety 2 (2014) 8-14 9 HAF © 2013 Vol. I, No. 2 ISSN: 2283-3927 1 Introduction Dermatophytoses, also called ringworm, are common contagious infections of keratinized tissues caused by keratinophilic/keratinolytic fungi named dermatophytes which affect a wide range of mammals, including man (Chermette et al. 2008). Man y species of dermatophyte have been reported as animal pathogens, bu t these may also be carried as normal flora on th e haircoat of pets (Cabanes et al. 1997; Cafarchia et al. 2006; Boyanowsky et al. 2000). Dermatophyte fungi can also be found in farm animals and roden ts (Cherm ette et al. 2008, Cabanes et al. 1997; Sargison et al. 2002; Mahmoud 1995; Moretti et al. 1998; Papini et al. 2009). There have been previous reports of isolation of dermatoph ytes from th e haircoat of health y carriers (Alteras et al. 1966; Chabasse et al. 1987, Marcianti et al. 1993) and cases of clinical dermatophytosis (Alteras et al. 1968, Knudtson et al. 1980, Peano et al. 2008) also in wild animals. Knudston and Robertstad (1970) dem onstrated a positive specim ens prevalen ce of 26.8% for keratinophilic fungi in 224 animals tested from 30 wildlife species in South Dakota. In a hair sample study in 2005 by Gallo et al. (2005), th e alpine marmot (Mar mota marmota) was identified as a reservoir for man y dermatoph ytes, including Microporum canis; and dermatophytosis has previously been reported in water buffalo (B ubalus bubalis ) (Pal et al. 1995). Clinical dermatoph ytosis has been reported in mule deer (Odocoileus hemionus ) (Wobeser et al. 1983), barking deer (Muntiacus muntjak) (Pa l and Thapa 1993) and Rusa deer (Cervus timorensis ) (Le Bec and Beugnet 1994). Conversely, in a recen t survey of fungal flora in white-tailed deer (Odocoileus virginia nus ) in Virginia (USA) no dermatoph ytes were identified (Hall et al. 2011). Rare studies of prevalence of dermatoph ytosis and ringworm episodes hav e also been signalled in wild animals in captivity (Kuntze et al. 1967, Janovitz and Long 1984). However, th e literature reporting the prevalence of dermatophyte carriers in the majority of wild species is still incomplete, and th e role of th ese species as reservoir for these pathog ens is unclear. The red deer (Cervus elaphus) is one of th e most represen ted animal throughou t the Alps. Measures have been tak en to preserve and manage thes e deer populations. Curren tly, nearly 10,000 red deer are present in th e in the Stelvio National Park (PNS) and adjacen t areas, of which around 60% liv e in a more or less stable community in the protected area that covers about 485 sq km. In 2000, th e Consortium of PNS started a project for the managem ent of deer populations in the Pa rk allowing, in some protected areas, controlled culling (reduction in numbers of populations by con trolled hunting) to reduce th e density of th e deer population, since high densities have significant im pacts on ecosystems and cause economic damage to agricultu ral activities. The project, however, has also provided a mechanism for investigation of the health status of th e deer and their relationship with the ecos ystem and with man. In this regard wildlife surveillan ce (to assess the presen ce of poten tially zoonotic pathogens on th e skin of wild deer) is an important area for which data is lacking. To our know ledge no studies report dermatoph ytes prevalence from red deer populations. The aims of this study were to determin e the prevalence, and identify species, of dermatophyte fungi in a population of wild alpin e red deer in Italy and to evaluate wh eth er asymptomatic deer may carry dermatoph yte fungi as part of the normal haircoat flora. Given the importance of the d eer population in the Stelvio National Park this knowledge is importan t in identifying the risk of human and livestock exposure to this zoonotic path ogen. R. Perego et al. - Int. J. of Health, Animal science a nd Food safety 2 (2014) 8-14 10 HAF © 2013 Vol. I, No. 2 ISSN: 2283-3927 2 Materials and methods Under a scientific project pursuant to art. 11, paragraph 4 of Law 6 December 1991 n. 394, and with permission of the Italian Ministry of Environm ent and Protection of Land and Sea (DPN 2010 – 13760, 17 june 201) and of the National Institute for Protection and Environmen tal Research (protocol n. 15387/T-A25, 6 may 2010), hair samples were collected from legally hunted red deer in Stelvio National Pa rk, Italy. The culling programm e provided a redu ction in the density of th e red deer population by allowing hunting within a small wintering area of about 1400 h ectares, comprising the unit of managemen t referred to as "Valfu rva-Sondalo", belonging to th e Forestry Station of Valfurva in the Lombard sector of PNS. This forestry station of about 24,600 hectares , the largest of the park, is of sufficient size to provide all th e needs of th e red deer (neighbourh oods for summering, wintering areas and breeding areas). Hair samples were collected between November and December 2012. All deer tested had been killed no more than 12 hours before sampling. All deer sampled were visually examined for dermatologic lesions. Samples obtain ed from 30 red deer consisted of 11 fawn s (5 females and 6 males) and 19 adult red deer (16 females, including 10 pregnan t females, and 3 males) ranging in age from yearling deer to mature deer (20 years). Although several meth ods are available for dermatoph yte testing, including Wood’s lam p examination, direct microscopic examination of hairs and fungal culture is considered the gold standard (Moriello 2003 ) and was the m ethod used in this study. The deer coat was brushed using a modified Mackenzie collection method (MacK enzie 1963; Moriello 2001). Briefly, a new sterilized toothbrush was used to collect hair samples by brushing each deer for a minimum of 2 minutes (at least 30 strok es) ov er the face, neck , abdomen, thorax and limbs (Hall et al. 2011) and immediately after brushing, all accumulated material was transferred to a sterile pou ch until it could be examined in the laboratory. In th e laboratory, a small sample of hair was collected from th e toothbrush with sterile, rubber - covered, hemostats and examined under a microscope (direct hair examination). Th e hairs were positioned in th e same orientation on a microscope slide, suspended in mineral oil, an d examined, with th e low-pow er objective of the microscope, for dermatoph yte hyphae an d arthrospores. 20% potassium hydroxide diaphanisation for 30 minutes of hair sampled was finally performed to microscopically detect ectotrix spores of Trichop hyton verrucosum. The coat samples w ere th en subjected to fungal cultu re: sample toothbrushes w ere gen tly imprinted onto th e surface of 9 cm Petri dishes con taining Sabou raud dextrose agar (with chloramph enicol 0.5% and actidione 0.4% added) on one half and Dermatophytes Tes t Mediu m (DTM) agar on the other half. An aliquot of samples was cultured on Sabouraud dextrose agar supplemented with inositol and thiamine to obtain more easily the possible grow th of Trichophyton verrucosum, a dermatophyte difficult to cultivate in normal dermatoph yte media (Peano et al. 2008). The Petri dishes were incubated upside down in two laboratory oven (MICRA 9T, I.S.Co Srl, Pieve Emanu ele, Italy) in th e dark, both at a constant temperature of 25 °C and 37° C, and examined daily for 3 w eeks. After 3 weeks (4 weeks for dishes at 37 °C), th e dermatophyte colonies on th e medium were macroscopically and microscopically examined and identified to species level. Macroscopic and microscopic examinations were also used to identify saprophytic fungi and yeas ts, but only to th e g enus level. F or each sample with R. Perego et al. - Int. J. of Health, Animal science a nd Food safety 2 (2014) 8-14 11 HAF © 2013 Vol. I, No. 2 ISSN: 2283-3927 saprophytic g rowth, the three predominant colony types w ere selected for saprophyte identification. 3 Results No deer included in this study presen ted with cutaneous lesions oth er than those inflicted by gunshot. No dermatophyte spores or h yphae were identified in an y sample using direct hair examination and no abnormalities (at the level of the bulb, shaft or tip) w ere identified in an y hair samples. DTM colou r change did not occur with any sample of cultu red hair and no dermatophyte colonies were grown. Only saproph ytic fungi were g rown - predominantly Alternaria spp., Mucor spp., and Cladosporium spp. (Table I) Samples from three subjects (10%) showed no fungal colon y g rowth, while two or more colonies were g rown in samples from 15 subjects (50%). Table 1 Saprophytic fungi isolated alone or togeth er on the sam e subject in th e study deer population. Th e isolation could be pure or a deer could have more of a saproph yte . Genus Number of isolation % Penicillum spp. 4 9.1 Alternaria spp. 13 29.5 Mucor spp. 10 22.7 Cladosporium spp. 7 16.0 Fusarium spp. 6 13.6 Candida spp. 4 9.1 Total 44 100 4 Discussion This is the first study of the presence and prevalence of fungi on the haircoat of wild red deer and one of the first on the fungal flora carried ou t on wild deer. The results of our study did not reveal the presence of asymptomatic carriers of dermatophy tes in th e red deer sampled population of Stelvio National Pa rk; in the investigated geographical area seem s therefore unlikely that the red deer act as a source of dermatophyte transmission to other wild animals, livestock or people with whom th ey migh t com e into contact. Due to th e absence of previous studies it is difficult to compare the results of this study with published literatu re. In a study published in 2013 by Nemeth et al. on th e inciden ce, clinical manifes tations an d demograph y of bacterial and parasitic dermatological diseases in white-tailed deer in th e southeastern United States an incidence of 5.7% of fungi (n.5 subjects on 88 deer with positiv e R. Perego et al. - Int. J. of Health, Animal science a nd Food safety 2 (2014) 8-14 12 HAF © 2013 Vol. I, No. 2 ISSN: 2283-3927 isolation) was reported. How ever, the study did not specify wh eth er the fungal isolates were dermatophytes, or wh ether isolations came from asymptomatic animals or th ose with dermatologic lesions. Th e on ly study of prevalence previously reported by Hall et al. in 2011 studied 60 adult white- tailed deer in Virginia (USA) but failed to isolate an y dermatophytes. . In our study fawns w ere well represen ted and th ere have been an ecdotal reports of outbreaks of dermatophytosis caused by fawns (Hall et al. 2011). Young animals are much more susceptible to infection, probably due to their naive immune system (Stannard and White 2002). Nevertheless, all th e fawns examined in our study, w ere n egative for dermatophytes by culture. The results of ou r study may be influ enced by th e sample group, g eographic limitations and season. The sample group consisted of only 30 animals. This low number of animals cannot be truly representative of the en tire deer population in th e park, (nearly 10,000 reed deer) notwithstanding the fact that th ere should have been a random selection of animals killed by hunters and the study group had a good representation of both sexes and the various age groups. All red deers were from a limited geographic region and therefore represen t a small subset of the Italian red deer population. Finally the deer w ere sampled in the late fall/early winter, during cooler weather, whereas dermatophyte flora is lik ely to be more prevalen t in hot, humid weather (Stannard and White 2002). Deer culling in the Stelvio National Park is always done in th e fall/ winter season because at this tim e th e deer population is closer to th e more accessible areas of the park wh ere the rangers place supplem entary sources of food. In calves of domes tic cattle, dermatoph ytosis is usually a self-limiting diseas e with a maximum duration of 4 m onths (Stannard and White 2002). It is possible that in th e red deer population outbreaks occur in fawns during spring and summer and resolv e by the fall. The saproph ytic fungi isolated from th e haircoat of 90% of th e red deer in this study were similar to those reported in the USA on white- tailed deer (Hall et al. 2011), showing that th e different g eographic area does not affect the commensal flora of the wild deer. 5 Conclusion In conclusion, studies on a larger sample of red deer, or with hair samples taken in other seasons, would be needed to confirm that red deer population from the Stelvio National Park doesn’t act as a reservoir for dermatoph ytes in this area . 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