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		<title>AAVS_Nexus 530</title>
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		<p>&nbsp;</p>
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		  <p class="Type-of-Article"><span class="CharOverride-1">Research Article</span></p>
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			<p class="title- ParaOverride-1">&nbsp;</p>
			<p class="title- ParaOverride-1">Clinical Study on Dermatophytosis in Calves with in vitro Evaluation of Antifungal Activity of Bergamot oil  </p>
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			<p class="Authors">&nbsp;</p>
			<p class="Authors"><span class="CharOverride-3">Wagdy Rady El-Ashmawy</span><span class="CharOverride-4">1</span><span class="CharOverride-3">, Ekbal Abd EL Hafez</span><span class="CharOverride-4">2</span><span class="CharOverride-3">, Haithem Abd El Saeed</span><span class="CharOverride-4">1</span></p>
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			<p class="Affiliations"><span class="CharOverride-5">1</span>Faculty of Veterinary Medicine, Cairo University, Giza, Egypt; <span class="CharOverride-5">2</span>Microbiology Department National Organization of Drug Control and Research, Egypt (NODCAR). </p>
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			<p class="Abstract ParaOverride-1">&nbsp;</p>
		  <p class="Abstract ParaOverride-1"><span class="CharOverride-6">Abstract </span>| Ringworm is a fungal and zoonotic infectious disease, caused by different species of dermatophytes. In this study, skin scrapings and hair samples were collected from beef calves recently introduced into a beef farm they have clinical signs of dermatophytosis. The collected samples were directly examined for fungal elements by direct microscopy and fungal culture. Fungal culture revealed <span class="CharOverride-7">Trichophyton verrucosum</span>. The antifungal activity of <span class="CharOverride-7">Bergamot oil</span> (<span class="CharOverride-7">Citrus Bergamia</span>) alone or in combination with salicylic acid using different concentrations was evaluated <span class="CharOverride-7">in vitro</span> and revealed that <span class="CharOverride-7">Bergamot oil</span> with different dilutions (1.25%, 2.5% and 5%) has a very effective antifungal effect against <span class="CharOverride-7">Trichophyton verrucosum</span>. </p>
			<p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-6">Keywords </span>| <span class="CharOverride-7">Bergamot oil</span>, <span class="CharOverride-7">Trichophyton verrucosum</span>, Antifungal activity, Dermatophytes, Beef calves.</p>
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			<p class="Editor----Citation">&nbsp;</p>
			<p class="Editor----Citation"><span class="CharOverride-9">Editor</span> | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.</p>
			<p class="Editor----Citation"><span class="CharOverride-6">Received</span> | October 11, 2014; <span class="CharOverride-6">Revised</span> | November 26, 2014; <span class="CharOverride-6">Accepted</span> | November 27, 2014; <span class="CharOverride-6">Published</span> | December 16, 2014&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-6">*Correspondence</span> | Wagdy Rady<span class="CharOverride-11"> </span>El-Ashmawy, Cairo University, Giza, Egypt; <span class="CharOverride-6">Email:</span> ubiowagdy@staff.cu.edu.eg</p>
			<p class="Editor----Citation"><span class="CharOverride-6">Citation </span>| El-Ashmawy WR, Abd EL Hafez A, Abd El Saeed H (2015). Clinical study on dermatophytosis in calves with in vitro evaluation of antifungal activity of Bergamot oil. Adv. Anim. Vet. Sci. 3(1): 34-39.  </p>
			<p class="Editor----Citation"><span class="CharOverride-9">DOI</span><span class="CharOverride-10"> | </span><a href="http://dx.doi.org/10.14737/journal.aavs/2015/3.1.34.39"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2015/3.1.34.39</span></a></p>
			<p class="Editor----Citation"><span class="Editor---Citation CharOverride-6" lang="en-US">ISSN (Online)</span> | 2307-8316; <span class="Editor---Citation CharOverride-6" lang="en-US">ISSN (Print)   </span> | 2309-3331</p>
			<p class="Editor----Citation"><span class="CharOverride-9">Copyright </span>© 2015 El-Ashmawy et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
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			<p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-1--Introduction----">INTRODUCTION</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Caps-on-First-Para ParaOverride-1"><span class="_idGenDropcap-1">R</span>ingworm in cattle is a highly contagious skin infection all over the world. It is an infection of the superficial, keratinized structures of the skin and hair of man and animals. The disease is caused by a group of keratinophilic filamentous fungi called dermatophytes in the Genera Trichophyton, Microsporum and Epidermophyton (<a href="#Gudding-R--Lund-A--1995-"><span class="Hyperlink">Gudding and Lund 1995</span></a>).<span class="CharOverride-7"> Trichophyton verrucosum </span>is the most common etiologic agent of cattle (<a href="#El-diasty-EM--Ahmed-MA--Okasha-N--Mansour-...-2013"><span class="Hyperlink">El-Diasty et al., 2013</span></a>).  </p>
		  <p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Animals are infected by contact with arthrospores (asexual spores formed in the hyphae of the parasitic stage) or conidia (sexual or asexual spores formed in the “free living” environmental stage). Transmission between hosts usually occurs by direct contact with a symptomatic or asymptomatic host <span class="CharOverride-6">(</span><a href="#Murray-PR--Rosenthal-KS--Pfaller-MA--2005-."><span class="Hyperlink">Murray et al., 2005</span></a>). It had been reported that housing animals in close proximity to each other for long periods in the presence of infected debris was responsible for the high incidence of the disease in winter (<a href="#Al-Ani-FK--Younes-FA--Al-Rawashdeh-OF--2002"><span class="Hyperlink">Al-Ani et al., 2002</span></a>). However, the disease appears to be more common in tropical than temperate climates, and particularly in countries or areas having hot and humid climatic conditions (<a href="#Radostits-OM--Blood-DC--Gay-CC--1997"><span class="Hyperlink">Radostits et al., 2007</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The  typical  lesion  in  cattle  presenting 10 to 50 mm patches of hair loss, desquamation and crust formation usually confined to the head, neck and, sometimes, other parts of the body surface is a heavy, greyish-white crust  that  is  raised  perceptibly  above  the  skin,  affecting young  calves’ more  than  adult  cattle  (<a href="#Cam-Y--G-m-ssoy-KS--Kibar-M--Apaydin-N--Atalay-O--2007"><span class="Hyperlink">Cam  et  al., 2007</span></a>;<span class="CharOverride-6"> </span><a href="#Akbarmehr-J--2011"><span class="Hyperlink">Akbarmehr, 2011</span></a>). Cattle ringworm is rapidly spread in the herd via infected propagules (hyphae and specialized fungal spores named arthrospores). The disease is responsible for great economic losses due to skin injuries and many casualties in animal products (wool, meat, etc.) as reported by <a href="#Weber-A--2000-"><span class="Hyperlink">Weber (2000)</span></a>. Dermatophytosis had been considered the most common zoonosis worldwide, affecting more children than adults (<a href="#Achterman-RR--White-TC--2012-."><span class="Hyperlink CharOverride-14" lang="en-US">Achterman and White, 2012</span></a>;<span class="CharOverride-6"> </span><a href="#Adeleke-SI--Usman-B--Ihesiulor-G--2008-."><span class="Hyperlink">Adeleke et al., 2008</span></a>). </p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">MATERIALS AND METHODS</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Animals in the Study</p>
			<p class="Body-Text ParaOverride-1">The study was carried out from September 2013 till 2014 (Autumn and Winter) in a beef farm at Giza governorate. The farm contains 250 beef calves (local mixed breeds) of 6-9 months age. Skin lesions appeared on 30 animals after 2 weeks of receiving calves.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Skin lesions also appeared on hands and face of 2 workers in the farm in contact with diseased animals. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Samples Collection</p>
			<p class="Body-Text ParaOverride-1">The samples of skin scraping and hair were collected from the periphery of the lesion after cleaning with 70% ethyl alcohol in sterile falcons. Samples were collected from infected animal’s suffering from lesions suggesting ring worm infections (Circumscribed areas of hair loss filled with raised white scales on head, neck or all over the body). <span lang="en-US">The study aims to isolate and identify the causative agent of ring worm from the affected calves and to evaluate the antifungal activity of </span><span class="CharOverride-7" lang="en-US">Bergamot oil</span><span lang="en-US"> </span><span class="CharOverride-7" lang="en-US">in vitro</span><span lang="en-US">.</span></p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Examination of Samples for Fungi</p>
		  <p class="Heading-2--History-in-MM-">&nbsp;</p>
			<p class="Heading-3">Direct Microscopical Examination using KOH (20%)</p>
			<p class="Body-Text ParaOverride-1">One or two drops of 20% KOH (potassium hydroxide) were placed on a microscopic slide and a small amount of the specimen was added and then, the slide was gently passed through a low flame and covered by a cover slip. After 2 h, the specimen was examined for the presence of arthrospores and hyphae under a light microscope according to <a href="#Ellis-D--Davis-S--Alexiou-H--Handke-R--Bartley-R--2007-."><span class="Hyperlink">Ellis et al., 2007</span></a>. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-3">Isolation and Identification of Dermatophytes from Samples</p>
			<p class="Body-Text ParaOverride-1">Scrapings of skin and hair were reduced in size to pieces approximately 1 mm across and the hair roots were cut into similarly sized fragments. Each sample was cultured on the surface of Sabaroude Dextrose Agar (SDA) containing chloramphenicol. The culture media were incubated at 25°C and 30°C for up to 5 and 21 days. After isolation the cultures were transferred to freshly prepared SDA media to obtain pure cultures. Pure cultures were also maintained in SDA slants at 5±1°C. The obtained dermatophytes were identified by their cultural morphology and microscopic characteristics. Microscopic identification of positive fungal cultures was carried out using the method described by <a href="#Murray-PR--Rosenthal-KS--Pfaller-MA--2005-."><span class="Hyperlink">Murray et al. (2005)</span></a>; <a href="#Chessesbrough-M--2006-"><span class="Hyperlink">Chessesbrough (2006)</span></a>. Briefly, a drop of lactophenol cotton blue stain was placed on a clean glass slide. A portion of mycelium was transferred into the lactophenol cotton blue stain and teased with a 22 gauge nichrome needle to separate the filaments. Cover slip was placed on the preparation and examined under low and high power magnification using a much reduced light for identification.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-3">Identification of Dermatophytes</p>
			<p class="Body-Text ParaOverride-1">The identification was based on colonial appearance, pigment production and the micro morphology of the spores produced. Cultures were examined at 4 or 5 days intervals from the onset. Some characteristics were also noted on the texture, colour and shape of the upper thallus and the production of pigment on the underside as described by <a href="#Ellis-D--Davis-S--Alexiou-H--Handke-R--Bartley-R--2007-."><span class="Hyperlink">Ellis et al. (2007</span></a><span class="Hyperlink">)</span>;<span class="CharOverride-6"> </span><a href="#Cowen-P--1990-.-M"><span class="Hyperlink">Cowen, (1990</span></a><span class="Hyperlink">)</span><span class="CharOverride-6">; </span><a href="#Rohde-B--Hartmann-G--1980-"><span class="Hyperlink">Rohde and Hartmann (1980</span></a><span class="Hyperlink">)</span>.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-"><span class="CharOverride-2">In vitro</span> Antifungal Assays</p>
			<p class="Body-Text ParaOverride-1">The following procedure was used according to <a href="#Magaldi-S--Essayag-S--Capriles-C--Perez-C--Colella-MT--Olaizola-C--Ontiveros-Y--2004"><span class="Hyperlink">Magaldi et al. (2004)</span></a>. Fungal spores were harvested after 7 days old on SDA slant. Culture was washed with 10 ml normal saline in 2% Tween 80 with the aid of glass beads to help in the dispersion of the spores. The spore suspensions were standardized to 10<span class="CharOverride-5">5</span> spores/ml. SDA was prepared according to specifications, autoclaved at (121°C for 15 minutes) and supplemented with 0.05% chloramphenicol and dispensed into 11 cm diameter Petri dishes. One ml of each standardized spore suspension (10<span class="CharOverride-5">5</span> spores/ml) was evenly spread on the surface of the SDA plates. Then, by using a special instrument known as sterile cork porer (6 mm in diameter), wells were made on the surface of SDA plates. Using Vernier callipers for measuring zone of inhibition.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Determination of Minimum Inhibitory Concentrations (MIC)</p>
			<p class="Body-Text ParaOverride-1">MICs for the <span class="CharOverride-7">Bergamot oil</span> were established using an agar dilution method (<a href="#Banes-Marshall-L--Cawley-P--Phillips-CA--2001"><span class="Hyperlink">Banes-Marshall et al., 2001</span></a>). Before the addition of the oils, 0, 5% (v/v) of Tween 20 was first added to the agar. Use of Tween 20, an emulsifier, enhances the solubility of oils in agar up to a final concentration of 4% (v/v) with oil, although at concentrations higher than this the oil will not solubilize even with Tween 20 incorporated into the agar. Initial experiments demonstrated that the incorporation of Tween 20 only into the agar had no effect on the growth of the bacteria under test.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Therefore controls had no oil nor Tween incorporated in the agar.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-3">Test Compounds and Susceptibility Testing Assays</p>
			<p class="Body-Text ParaOverride-1">Natural essence of bergamot produced by the Consorzio del Bergamotto of Reggio Calabria, Italy, were supplied by Bergamon S. r. l. (Rome, Italy).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-3">Mold inoculum preparation</p>
			<p class="Body-Text ParaOverride-1">All molds were subcultured from stock cultures onto plates of either Sabaroude dextrose. These plates were then incubated at 30°C until a lawn had developed over the entire plate. The inoculums were prepared by aseptically cutting the lawn growth into 10x10 mm squares.</p>
			
			<p class="Heading-3">&nbsp;</p>
		  <p class="Heading-3">Plate preparation and analysis</p>
			<p class="Body-Text ParaOverride-1">The agar dilution method described above was modified for filamentous fungi by substituting either malt extract or Sabauroud dextrose agar as the test medium and incubating cultures at 30°C until growth of the positive control covered approximately 90% of the agar plate.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The MIC was recorded as the lowest concentration of test material where at least five out of the six readings showed no growth. Growth of less than 5 mm around the inoculums was considered a negative result. Growth of greater than 5<span class="CharOverride-2"> </span>mm around the lawn was considered a positive result.</p>
			
			<p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-1--Introduction----">Results</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The study was carried out during September 2013 till April 2014 (Autumn and Winter months) in a farm containing 250 beef calves (local mixed breeds) aged 6-9 months. Clinical signs of skin lesions appeared on 30 animals after 2 weeks of receiving calves in the form of Circumscribed areas of hair loss filled with raised white scales on head, neck or all over the body (<a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>, <a href="#Figure-2"><span class="Hyperlink">2</span></a> and <a href="#Figure-3"><span class="Hyperlink">3</span></a>) also skin lesions also appeared on hands and face of 2 workers in the farm in contact with diseased animals. All examined samples were positive for fungal elements (ectothrix spores and endothrix hyphae) by direct microscopic examination. The fungus grew slowly on SDA producing white, cottony, heaped and slightly folded colonies with some submerge growth and yellow reverse pigment (<a href="#Figure-4"><span class="Hyperlink">Figure 4</span></a>). Microscopic examination  of  isolates  stained  with  lactophenol  cotton  blue revealed  septate  hyphae  with  numerous  clavate  microconidia borne laterally from the hyphae and many  clamydospores  arranged  in  chains  (chains  of pearls) characteristic of <span class="CharOverride-7">T. verrucosum</span>.</p>
			<p class="Body-Text ParaOverride-1">&nbsp; </p>
            
            <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150105200043.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150105200043.png" width="80" height="80"></a>
    <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-1-"></a>Figure 1:</span> Ring worm lesions of face, neck and body of a calf.</p></div><br>
    
    
    <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150105204749.jpg" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150105204749.jpg" width="80" height="80"></a>
   	  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-2"></a>Figure 2: </span>Typical ring worm lesions on the body of a calf</p></div><br>
            
            <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150105204850.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150105204850.png" width="80" height="80"></a>
           <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-3"></a>Figure 3:</span> Typical ring worm lesions on the head of a calf</p></div><br>
           
    
    <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150105201051.jpg" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150105201051.jpg" width="80" height="80"></a>
   <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-4"></a>Figure 4:</span> <span class="CharOverride-7">Trichophyton verrucosum</span> growth on sabaroude dextrose agar</p></div><br>
   
  
  <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150105204751.jpg" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150105204751.jpg" width="80" height="80"></a>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-5-"></a>Figure 5:</span> <span class="CharOverride-7">Trichophyton verrucosum</span> grow on potato dextrose agar and shown antifungal activity bergamot 1.25% with zone of inhibition 28 mm</p></div><br>
            
          
          <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150105200653.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150105200653.png" width="80" height="80"></a>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-6-"></a>Figure 6:</span> <span class="CharOverride-7">Trichophyton verrucosum</span> grow on potato dextrose agar and shown antifungal activity bergamot 2.5% with zone of inhibition 32 mm</p></div><br>
            
           <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150105205153.jpg" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150105205153.jpg" width="80" height="80"></a>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-7--T"></a>Figure 7:</span> <span class="CharOverride-7">Trichophyton verrucosum</span> grow on potato dextrose agar and shown antifungal activity bergamot 5% with zone of inhibition 40 mm</p></div><br>
            
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Antimicrobial Activity of <span class="CharOverride-2">Bergamot oil</span></p>
			<p class="Body-Text ParaOverride-1">The <span class="CharOverride-7">Bergamot oil</span> has antifungal activity against a <span class="CharOverride-7">Trichophyton verrucosum</span> in oil concentration 1.25%, 2.5% and 5% as a zone of inhibition of 28mm, 32mm and 40 mm in <a href="#Figure-5-"><span class="Hyperlink">figure 5</span></a>, <a href="#Figure-6-"><span class="Hyperlink">6</span></a> and <a href="#Figure-7--T"><span class="Hyperlink">7</span></a>, respectively.</p>
			
			
			<p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-1--Introduction----">&nbsp;</p>
		  <p class="Heading-1--Introduction----">Disscussion</p>
			<p class="Body-Text">&nbsp;</p>
			<p class="Body-Text">Dermatophytosis is one of the economically important fungal disease of animals and has a zoonotic importance. The study was carried out in a beef farm at Giza governorate during the period between September 2013 and April 2014. After 2 weeks of receiving the calves aged 6-9 months skin lesions appeared on 30 calves out of 250 (12%). Lesions appeared in the form of circumscribed area of alopecia filled with white scales on face, head neck or all over the body. <span class="CharOverride-7">Trichophyton verrucosum</span> was the only fungus isolated from affected the examined calves. This indicates that <span class="CharOverride-7">T. verrucosum</span> is the main fungus causing ring worm in cattle as reported by <a href="#Dalis-JS--Kazeem-HM--Kwaga-JKP--Kwanashie-CN--2014"><span class="Hyperlink">Dalis et al., 2014</span></a>. The dermatophytosis may be a major problem in calves specially under stress conditions like transportation and changing the place and managemental conditions as in the farm under study and that agree with previous findings of <a href="#Dalis-JS--Kazeem-HM--Kwaga-JKP--Kwanashie-CN--2014"><span class="Hyperlink">Dalis et al. (2014)</span></a>;<span class="CharOverride-6"> </span><a href="#Cam-Y--G-m-ssoy-KS--Kibar-M--Apaydin-N--Atalay-O--2007"><span class="Hyperlink">Cam et al. (2007)</span></a>;<span class="CharOverride-6"> </span><a href="#Shams-Gahfarokhi-M--Mosleh-TF--Ranjbar-BS--Razzaghi-AM--2009"><span class="Hyperlink">Shams-Gahfarokhi et al. (2009)</span></a> that young animals are particularly susceptible to infection by ringworm  </p>
			
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">fungi. This could be as a result of the poorly developed immune system and the high pH of the skin in young animals (<a href="#Radostits-OM--Blood-DC--Gay-CC--1997"><span class="Hyperlink">Radostits </span><span class="Hyperlink">et al., 2007</span></a>). Diagnosis of ringworm in this study was based on clinical signs, demonstration of fungal elements in samples by direct microscopic examination and the isolation of causative agent by culture. <span class="CharOverride-7">T. verrucosum</span> had been implicated in ringworm of cattle (<a href="#Dalis-JS--Kazeem-HM--Kwaga-JKP--Kwanashie-CN--2014"><span class="Hyperlink">Dalis et al. (2014)</span></a>;<span class="CharOverride-6"> </span><a href="#Swai-ES--Sanka-PN--2012"><span class="Hyperlink">Swai and Sanka, 2012</span></a>;<span class="CharOverride-6"> </span><a href="#Cam-Y--G-m-ssoy-KS--Kibar-M--Apaydin-N--Atalay-O--2007"><span class="Hyperlink">Cam et al., 2007</span></a>). The main clinical signs observed among the affected calves were circular, circumscribed, grayish-white, thick crusty lesions perceptibly raised above the skin. The lesions were most frequently found on the head and neck especially around the eyes and face. These observations were in agreement with other reports of <a href="#Dalis-JS--Kazeem-HM--Kwaga-JKP--Kwanashie-CN--2014"><span class="Hyperlink">Dalis et al. (2014)</span></a>;<span class="CharOverride-6"> </span><a href="#Akbarmehr-J--2011"><span class="Hyperlink">Akbarmehr (2011)</span></a>; <a href="#Cam-Y--G-m-ssoy-KS--Kibar-M--Apaydin-N--Atalay-O--2007"><span class="Hyperlink">Cam et al. (2007)</span></a>. The reason for the occurrence of more lesions around the eyes and face in young animals is not well understood. However, the habit of licking and grooming by calves could predispose this part of the animal to infection. Our results  showed  that  all  the  samples  examined  by direct  microscopy  were  positive  for  fungi. Other researchers found out that direct microscopic examination could provide a positive diagnosis in 60-71% of samples from which dermatophytes were isolated as in case of <a href="#Dalis-JS--Kazeem-HM--Kwaga-JKP--Kwanashie-CN--2014"><span class="Hyperlink">Dalis et al. (2014)</span></a>;<span class="CharOverride-6"> </span><a href="#Al-Ani-FK--Younes-FA--Al-Rawashdeh-OF--2002"><span class="Hyperlink">Al-Ani et al. (2002)</span></a>;<span class="CharOverride-6"> </span><a href="#Sparkes-AH--Gruffy-TJ--Shaw-SE--Wright-AI--Stokes-CR--1993-."><span class="Hyperlink">Sparkes et al. (1993)</span></a>. In this study, the dermatophyte was isolated in pure culture suggesting that SDA is a suitable selective medium for the isolation of pathogenic fungi. Colonies of <span class="CharOverride-7">T. verrucosum</span> in this report were slow growing, white, cottony, heaped and slightly folded with some submerged growth and yellow reverse pigment. This observation is consistent with the findings of <a href="#Dalis-JS--Kazeem-HM--Kwaga-JKP--Kwanashie-CN--2014"><span class="Hyperlink">Dalis et al. (2014)</span></a> and <a href="#Forbes-BA--Sahm-DF--Weissfeld-AS--2002-."><span class="Hyperlink">Forbes et al. (2002)</span></a>.  </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The warm and humid climate of our environment could have favoured the growth and development of fungal spores thereby predisposing the animals to infection and hence the outbreak in this highly susceptible population. Cattle  ringworm  causes  high  economic  losses  especially  in  the  livestock  and  leather  industries  due  to downgrading of hides  and skin and decrease in meat and milk production vv as mentioned by <a href="#Gudding-R--Lund-A--1995-"><span class="Hyperlink">Gudding and Lund (1995</span></a><span class="Hyperlink">)</span>. The major problem with <span class="CharOverride-7">T. verrucosum</span> infection in cattle farms is that, once the disease is introduced into a farm, it spreads rapidly among susceptible animals. The organism  is  difficult  to  eradicate  from  the  environment  because  of  the  peculiarity  in  composition  of  its  spores.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Treatment of cattle ringworm is expensive and cumbersome  especially  on  a  herd  level  as mentioned by <a href="#Gudding-R--Lund-A--1995-"><span class="Hyperlink">Gudding and Lund (1995</span></a><span class="Hyperlink">)</span>, this may be due to difficult application of most antifungal drugs also they are expensive. In this study we evaluate the antifungal activity of <span class="CharOverride-7">Bergamot oil </span>(Citrus Bergamia) against <span class="CharOverride-7">Trichophyton verrucosum</span> in order to provide an effective cheap antifungal drug easy for application. In the study we use different concentrations of <span class="CharOverride-7">Bergamot oil</span> 1.25%, 2.5% and 5% and use it alone and in combination with salicylic acid. The study reported that <span class="CharOverride-7">Bergamot oil</span> has antifungal activity against <span class="CharOverride-7">Trichophyton verrucosum</span> at all concentrations (1.25%, 2.5% and 5%) with and without combination with salicylic acid. The study results give substantial support to popular or anecdotal beliefs in the effectiveness of treating skin with <span class="CharOverride-7">Bergamot oils</span>. Also, the Antimycotic action of <span class="CharOverride-7">Bergamot oil </span>especially on <span class="CharOverride-7">Trichophyton verrucosum</span> and we recommended the application <span class="CharOverride-7">in vivo</span> and determine the antifungal activity and the best method for application.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Conclusions</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">In the present study we could conclude that, <span class="CharOverride-7">Trichophyton verrucosum </span>is the main cause of ring worm in cattle and has an occupational hazard appeared mainly in young calves under stress conditions. The study also high lights the significant antifungal properties of <span class="CharOverride-7">bergamot oil</span>.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Consequently, in the field of elimination of ringworm infection, <span class="CharOverride-7">bergamot oil</span> could be proper candidates disinfectant agents and could be used as active ingredient for dermatological applications.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">ACKNOWLEDGEMENTS</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">We would like to thank Dr Shehab El Din Talat at NODCAR for his assistance in editing this research.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">References</p>
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