<4D6963726F736F667420576F7264202D20E32EE32E20C7D3EDE120C7C8D1C7E5EDE3> Al – Ameed Diyala Agricultural Sciences Journal, 5( 2 ) 1 – 13 ,2013 1 ISOLATION AND IDENTIFICATION OF FUNGI FROM INFECTED MILK SAMPLES OBTAINED FROM CATTLES WITH MASTITIS AND STUDING THE ANTIFUNGAL ACTIVITY OF ROSEMARY ETHANOLIC EXTRACT AGAINST THE MAIN STRAINS . Aseel. I. Al-Ameed * Assist. Lecturer - College of Vet. Med. zoonoses diseases unit.-Univ. of Baghdad. Republic of Iraq. aseel-alam @ yahoo.com. ABSTRACT Objective: The purpose of this study was to isolate and identification fungi from the samples of cows milk with mastitis, the samples were collected during the period between February- April 2009 in the Abu-Ghraib zone. Material: In the present study a 100 samples of mastitis cows milk was collected. Results: Eighty samples (80%) were showed positive fungal infections, of which 78.75% (63 samples) was yeast and 21.25% (17 samples) was molds. The main strains which were isolated and responsible about high percentage of infection were the followings, Candida albica ns25%. Geotrichumcandidum20%, Rhizopus sp p .17.5%, and other species which include, Candida spp.11.25%, Sacchromycescerevisiae11.25%, Candida tropicalis 7.5%, Crypto coccus neoformans2.5%, Pen icilliu m spp. 2.5%, Rodotorula spp. 1.25% and Aspergillus terreus1.25%, while 20 samples showed a negative results. Rosemary ethanolic extract showed a different antifungal activity aginst the main strains of isolates (C.albicans, Sacch. cerevisiae, Geot. candidum, Asp.terreus, and Rhizopusspp). There was an inhibition zone of using extract in a concentration of 200mg/ml on the growth of C.albica ns, Geot. candidumand Sacch. cerevisiae, while a concentration of l00mg/ml of the extract was effective only against Sacch.cerevisiae. The extract showed antifungal activity against the growth of Asp. terreusin all concentrations, with minimum inhibitory concentration (MIC) of l0mg/ml, in contrast, the extract had no effect on the growth of Rh izo pusspp. in low concentration (10, 20 mg/ml) and only a concentration of 40, 80 mg/ml showed a minimal effect compared with the antifungal drug (Clotrimazole). Conclusion: The presence of yeasts and molds in cows milk indicate that the mycotic mastitis may be occurred in association with bacterial mastitis. Rosemary ethanolic extract had an antifungal activity most probably due to the presence of some compounds (α-Pinene, Bornyl acetate, Camphor and 1,8- Cineole) that responsible about this property. Key Words: Samples of mastaticcows milk, Fungi, Rosemary ethanolic extract. ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Received for publication Feb. 15 , 2012 . Accepted for publication Dec. 31 , 2012 . Al – Ameed Diyala Agricultural Sciences Journal, 5( 2 ) 1 – 13 ,2013 2 INTRODUCTION Fungi are wide spread in nature, being noted in bedding and gear from the stables on milking machines. Mycotic mastitis also existed in cattle before the arrival of antibiotics, however, since then there has been an ever increasing number of cases reported, almost always associated with prior antibiotic treatment of suspected or proven bacterial mastitis (Lagneau etal .,1996). In most cases, bacteria are recognized as the primary pathogens while, fungi particularly yeasts, have been regarded as secondary invading pathogens of mastitis (Costa et al.,1998). Mycotic infections of the mammary gland usually occurs as sporadic cases affecting a small percentage of cows, or as outbreaks affecting the majority of animals. In both situations, however, the seriousness of infection depends on the number of organisms present in the glands and the species of yeast and mold involved (Farnsworth , 1977). Yeasts are a group of unicellular organisms, ever present in the natural surrounding of dairy cattle and are normal inhabitants of the skin of the udder and teats. Yeasts are considered as opportunistic pathogens which colonize the cows udder (Richared etal.,1980). The use and abuse of antibacterial drugs, treatment with contaminated antibiotic solutions, as well as syringes, or other materials brought in contact with the mammary gland may favor yeast colonization of cows udders(Stantos and Marin, 2005; Costa etal.,1998; Gibbony etal. , 1970). Different fungi have been reported as a cause of mycotic mastitis such as Aspergillusfumigatus , Aspergillusterreus, Candida spp., Cep h alo sp oriu m spp., Coccidioides spp., Cryptococcus neoformans , Geotrichumcandidium, Histoplasma spp., Mucor spp., Rhizopus spp., Torulopsis spp. and Trichosporon sp p . (Krukowski etal., 2000; Aalbaek etal., 1994). Furthermore other fungi such as Cryptococcus spp., Rh odo to rula spp., Trichosporumcutaneum, Aureobasidiumpullulansand pichiaohmeri, have also been isolated from the milk of healthy glands (Lagneau etal. , 1996; Costa etal., 1993). There is an increasing interest in phytochemicals as new sources of natural antioxidant and antimicrobial agents. The use of synthetic antioxidants in the food industry is severely restricted as to both application and level (Tawaha, 2007; Peng, 2005). Currently, there is a strong debate about the safety aspects of chemical preservatives, since they are considered responsible for many carcinogenic and teratogenic attributes, as well as residual toxicity (Moreira, 2005). Growth of microorganisms in food may cause spoilage or food borne disease (Delcampo etal. , 2000). Rosemary (Rosmarinusofficinalis) is of Lamiaceae (Labiatae) family is a spice and medicinal herb widely used around the world. Of the natural antioxidant, rosemary has been widely accepted as one of the spices with the highest antioxidant activity (Peng, 2005). Rosemary extracts and essential oil is also used an antibacterial, antifungal (Oluwatuyi, etal.2004; Fernandes-Lopez, etal.2004), and anticancer agent (Leal, 2003). High percent of the antimicrobial Al – Ameed Diyala Agricultural Sciences Journal, 5( 2 ) 1 – 13 ,2013 3 activity is attributed to carnosic acid and carnosol. It is clear that rosemary extracts have bioactive properties, but their antimicrobial activities have not been deeply characterized. Antimicrobial activities of plant essential oils have been known for centuries (Delcampo et al., 2000). The mechanism of action has not been studied in great detail considering the large number of different groups of chemical compounds present in essential oils. The main compounds responsible for the antimicrobial activity are α- Pinene, Bornyl acetate, Camphor and 1,8-Cineole (Daferea, et a.l2000). The aim of this study was to isolate and identify" fungi (yeast and molds) from cows milk with mastitis in veterinarian clinics of Abu-Ghraib zone and to investigate antifungal activities of rosemary ethanol extracts on the main and important isolates and to detect the minimum inhibitory concentration(MIC) value of the extract. MATERIALS AND METHODS Milk samples: One hundred milk samples from quarters with clinical and subclinical mastitis were examined. The samples were collected between February- April 2009 from mastitis cows in veterinarian clinics of Abu- Ghraib zone and these samples were investigated in laboratories of zoonosis diseases unit in the Vet. Med. College /Baghdad University. The milk samples were always taken aseptically, kept at temperature of 4C° and plated, at the latest 24 h. after sampling. Isolation of fungi: Milk samples were also inoculated on Sabouraud dextrose agar (SDA) with chloramphenicol (0.05mg/ml), which then incubated at 28-30 C° for 2days-3weeks. The isolated fungi were classified according to the colony characteristic, microscopic examination after staining with lactophenol cotton blue stain (LPCB) in a wet mount, hyphae's size and shape of spores were noticed. The identification of the yeast growth based on the morphological colony, physiological and biochemical characteristic, by using Gram stain for staining, ability to growth at 37C° and 42C°, germ tube test, urease production, presence of capsule, caffic acid ferric citrate agar test, and carbohydrate fermentation tests for detection of Candida spp., Cryptococcus neoformans and other species of yeasts were also under taken. Rosemary extract: The leaves of rosemary ( Rosmarinusofficinalis) plant were obtained from the local market and verified by Iraq National Herb, Agricultural Ministry. The leaves were cleaned, dried and then ground to powder, about 100 gram of powder was suspended in 500 ml of 70% ethanol and kept on shaker at room temperature over night, then separated using separator funnel, subsequently filtered through whatman filter paper No.l and filtrate dried(Harborne, 1984), the dried extract was weighted to prepare the stock solution by dissolving (4g) of extract powder in 20 ml of distaled water with 5% dimethyl sulfoxide (DMSO) to prepare a concentration of 200mg/ml, from which another concentration (100,50) mg/ml were prepare for yeasts and another concentration Al – Ameed Diyala Agricultural Sciences Journal, 5( 2 ) 1 – 13 ,2013 4 (10, 20,40, 80) mg/ml for molds to determine the minimum inhibitory concentration (MIC) of the extracts on growth of yeasts and molds. Note: MIC was measured by the lower concentration of extract which gave inhibition to the growth of strains. Fungal culture: The antifungal activity of 70% ethanolic extract of rosemary was tested against (3) isolates of yeasts from milk samples (Candida albicans, Geotrichumcandidum, Saccharomyces cerevisiae) and (2) isolates of molds(Asp erg illu sterreu s and Rhizopusspp.), the antifungal effect of prepared extract was tested on SDA by using agar geldiffusion method in case of yeasts and agar dilution method by adding the extract in different concentration to the SDA media in case of molds, by adding (1,2,4,8) ml of stock solution (200 mg ml) of extract, to (19, 18, 16, 12) ml Of SDA, to obtained the different concentration (10, 20, 40, 80) mg/ml and inoculated the molds on the surface by cut 5 mm diameter of each mold and inoculate it at the center of plate then incubated at 28-30 C° for 3-8 days. Diameter of growth inhibition zone for different molds were measured for each one in duplicate average of two perpendicular diameter and according to this equivalent (Lima etal., 1992). Compared with the control plate. Inhibition% = [(C-T)/C] x 100 C=the colony diameter of the mycelium on the control petridish. T=the colony diameter of the mycelium on the test petridish. Agar gel diffusion method:- The 3 isolates of yeasts cultured on a sabouraud dextrose broth and incubated at 25-30 C° for 48 hrs. the inoculums was standardized according Mcfarlands turbidity standard. The turbidity was compared with mcfarlands 0.5 standard which provide turbidity comparable to yeast suspension containing 1.5x108CFU/ml (Lima etal., 1995). This test was performed using the standard procedure as described by (20) the inoculums suspension of each yeast strains was swabbed on the entire surface SDA, holes of 7mm in diameter were made with stainless steel cylinders and filled (60µl) microliter with the fluid extract in different concentration (200,100,50) and fourth hole filled with distal water as control, then left at ambient temperature for 15min. to allow excess per diffusion of extract prior to incubation at 25-30Cο for 48-72 hrs. Inhibition zones were measured and expressed in mm by notice the inhibited growth around the holes compared with the control plate. At that time the antifungal impacts of Rosemary ethanolic extract compared with Clotrimazole antibiotic (0.25 mg/ml)for yeasts and molds was studied. RESULTS AND DISSCUSION A total of 100 samples of cows milk with mastitis was studied mycologically for pathogenic fungi, from which 80 samples were positive for fungal infections and a different species of yeasts and molds were isolated .(Table 1) Al – Ameed Diyala Agricultural Sciences Journal, 5( 2 ) 1 – 13 ,2013 5 C. albica ns was the major cause of infections 20(25%), then Geot. candidum16(20%), and R h izopusspp. 14(17.5%). While Candida spp. 9(11.25%), Sacch . cerevisiae9(11.25%) and C.tropicalis6(7.5%), and miner cause was Crypt. neoformans 2(2.5%), Penicillium spp . 2(2.5%), Rodotorulaspp. 1(1.25%) and Aspergillusterreus 1(1.25%), whereas 20 milk samples shows a negative results for fungal infection. The results revealed that the rosemary ethanolic extract showed an antifungal activity against the main pathogenic isolates from mastaticcows milk, (table 2) and the concentration of 200 mg/ml had the strongest effect by giving large zone of inhibition on Geot. candid iu m(19 mm), Sacch. cerevisiae(17mm), and small zone on C . albica ns (12mm), while at the Table 1:Species of fungi (yeasts and molds) .) isolated from mastatic cows milk. concentration of 100 mg/ml the inhibition zone was (11mm) in case of Sacch. cerevisiae, and the others ( C.albica ns , Geot.candidium) gave a negative results, whereas the concentration of 50 mg/ml had no effect on the three strains, compared with the antifungal drug (Clotrimazole) that give large inhibition zone in case of Sacch. cerevisiae(40mm), C.albicansand Geot. candidum(35mm). The zone of inhibition increased with increasing in concentration of ethanolic extract. Al – Ameed Diyala Agricultural Sciences Journal, 5( 2 ) 1 – 13 ,2013 6 Table (3) showed the effect ofethanolic extract of rosemary on molds (A.terreusand Rhizopusspp.), the MIC of 10 mg/ml concentration on A. terreusand the percentage of growth inhibition was 65%, whereas the concentration of 40 mg/ml record high percentage of growth inhibition 77.5 % compared with the effect of antifungal drug (Clotrimazole) 0.25mg/ml which give similar percent 79.4%. However, the conc. of 80 mg/ml of extract showed higher percentage 83.75% compared with Clotrimazole and at 20mg/ml which showed a low effect 71.25%. While there was no antifungal activity of the extract against Rhizopusspp. at the concentration 10,20 mg and shows low percentage of growth inhibition 11.8 %, 23.5% in the concentration 40, 10 mg/ml. and the MIC of it at 40mg/ml compared with the plate treated with Clotrimazole showed 83.5% compared with control plates. (MIC was measured by the lower concentration of extract which gave inhibition to the growth of strains). There is a reverse proportion between the mean of fungal growth and the concentration of extract, Al – Ameed Diyala Agricultural Sciences Journal, 5( 2 ) 1 – 13 ,2013 7 the fungal growth decreased in the diameters when the conc. increased, in contrast; the percen tage of growth inhibition was increased when the concentration increased. Mastitis is an infection of the cows udder, caused by micro- organisms freely encountered in the environment. Fungi are opportunistic microorganisms which parasitized the animals weakened immunological system and develop by causing disease and impairing th e normal flow of milk , with h eavy loss for the far m owner (Wawron and Szczubial, 2001). In this study, yeasts were isolated in a percent 78.75% of all samples analyzed, Candidaspp. were the predominant eukaryotes one isolated in 43.75%, and th e most important species of Candidawas gave th e highly percentage of infection was C.albicans(25%), while other types of Candidareported 11.25% , and C.tropicalis7.5%. the present study was accordant with (Lagneau, et al.1996; Santos and Marin, 2005; Williamson and Dimenna, 2007 and Barros et al.,2011) who were reported th at th e predomin ant yeasts cause bov in e mastitis are Candidaspp., and C.albicans was isolated in a highpercent. In several surveys of my cotic mastitis C.tropicaliswas the most important one (Richared etal.,1980). C.albicanshas been reported as the mo st co mmo n sp ecies o f yeast pathogen found and cause mastitis problems. Candidaspp. normally lives in saprobiosis, although in favorable circumstances, it may develop its pathogenic potential. As a rule, Candidaspp. occurs in milk withou t any associated pathogens, although it may cau se mastitis in th e su b-clinical, clinical or chronic modes (Wawron and Szczubial, 2001). High contamination index by Candida may be related to lack of hygiene during th e milking process. Geot.candidum is an opportunistic, keratin oph ilic y east-lik e fungus. In the past, there are only a very few reports from around the entire world regarding its incrimin ation with bo vin e mastitis. Mishra and Panda, 1986 found only one case (0.7%) of Geot.candidumcause mastitis. In contrast, Costa et al.(1993) detected (6.4 %) positive mastitis forGeot. candidum, and Chah ota et al.(2001) isolated itonly from clinical mastitis in co w, this stud y was agreement with the previous study in which was isolated this species in 20% from bovine mastitis. Another yeasts were repo rted in this study are Sacch. cerevisiae(11.25%), Crypt. neoformans(2.5%) and Rodotorulaspp. (1.25%).There are many regional differences in yeast species and their percentage of its causing ma stitis, Turkyilmaz and Kaynarca, 2010 have been reported Crypt.neoformans(2.4%) and Sacch. cerevisiae(2.4%), also Pengov, 20 02 showed that the Crypt. neoformansrevealed (2%) of isolated strain s, wh ile Co sta, etal.1 99 3 have been repo rted that the Cryptococcusspp. (71 strains), Rhodotorulaspp. (40 strain). Al – Ameed Diyala Agricultural Sciences Journal, 5( 2 ) 1 – 13 ,2013 8 The molds classified in following genera were also isolated in this Research Rhizopusspp.(17.4%) ,Penicilliumspp.(2.5%), and Asper.terreus(1.25%). Costa etal.(1993) recorded th at the molds were in (11.95%) classified in the following Aspergillus,Penicillium(1.2%) which similar to this study. In Iraq (1992) الذهيبات detected molds and yeasts In Abu- Gh raib zo n e in winter Aspergillus88%, Rhizopus20%, Penicillium12% from cow milk and yeasts Sacch. cerevisiae28%, Candida spp.20%, Rodotorula20 %. Th is is rev erse with th is stud y in wh ich th e Aspergillusshowed the high percent, while the Rhizopusan d Penicilliumrecord the lower, and Sacch.cerevisiaerecord the high p ercent wh ile Candida spp.and Rodotorulathe lower one. Results show that although fungi may peacefully live with th e host, found in a great variety of sub strates, su ch as ma mmary g land , hands, soil and water, they may develop their pathogenic power and cause infections when they encounter favorable cond ition s su ch as environmental contamination associated with lack of hygiene during the milking, poor equipment’s cleaning,some diseases that change the cell immunity and excessive or erratic use of antibioticsafter bacterial mastitis treatment which represented the ma in factor that propitiatebecause they affect the microflora of the mammary glan ds wh ich acts as an animal natu ral defense and th is agree with Aboul- Gabal, etal.1977. In addition, large doses of antibiotics may cause reduction in th e Vitamin A, leading to injury to the udders epithelium, facilitating the invasion of fungi (Krukowski et al., 2000). Adequate management procedures, especially concerning the milk process, and the hygiene methods employed at this stage of milk production, may lessen the occurrence of fungus produced mastitis and decrease its contamination during th e milking process. The results indicated th at th e ro semary extract showed antifungal activity agin st many strains, an d Geot. candidiumgave high inhibition zone then Sacch. cerevisiaeand the lower one is C.albicansat the concentration 200mg/ml and the MIC of Geot. candidum, C. albicanswere detected 200mg/ml, while in case of Sacch. cerevisiaewas l00mg/ml. However the plates treat ed with Clotrimzole gave high inhibition zone compared with plates which treated with extract. On the study by هادي (2007) showed antifu ngal activity of rosemary ethanolic extract on C.albicansdetect th e zo ne of inhibition 12mm at a concentratio n 150 mg /ml and th e MIC was 75mg/ml gave 9mm inhibition zone who agreement this study, while, the effectiveness of extract on mo ld s (A.terreusand Rhizopus)more over than yeasts . The gr owth inhibition of A.terreuswas in all concentrations and the MIC was l0mg /ml, in contrast, the Rhizopusspp. growth showed no inhibi tion on concentration 10, 20 Al – Ameed Diyala Agricultural Sciences Journal, 5( 2 ) 1 – 13 ,2013 9 mg /ml and the MIC was 40 mg /ml, Santoyo et al.(2005) found that the volatile oil of rosemary has inhibition effect on the growth of C.albicans and Asp .niger which agreement the present study. The results indicated that the rosemary ethanolic extract showed antifungal activity according to Moreno, etal. (2006) because contained many compounds which responsible for this property. And these compounds act by inhibiting cell wall synthesis of micro- organisms, decrease essential protein synthesis, composed of a complex which involved with the cell wall and retarded the permeability, inhibit enzymes metabolization which important in growth and reproduction and rupture the cell membranes or change the function of its (Cowan ,1999). Identification of yeasts and molds in cows milk samples were analyzed demonstrate the occurrence of mycotic mastitis which lead to decrease in milk quality, and consumption milk contaminated by fungi or their toxins from human being which lead to human fungal infections or diseases, early diagnosis of mastitis by using California mastitis test (CMI) in Iraqi farms, and antibacterial therapy with previous susceptibility tests may be recommended to decrease fungal infection or mycotic mastitis. CONCLUSION Cows milk may be obtained by human or it’s used in food industry like cream, cheese, Butter, …etc. or other industrials which involved milk with their industerlization so these product should be free from any contamination. Rosemary extracts may be promising with regard to their incorporation into various foods, pharmaceutical products and foods industry for which a natural aroma, colored and antioxidant/antimicrobial additive is desired. These properties are also needed by the food industry in order to find possible alternatives to synthetic preservatives. Further studies are necessary to investigate the incorporation of extracts in to appropriate food, antioxidant and antimicrobial activities in the whole food system. 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J.55: 188- 9 0 . دراسة و خوذة من التهاب الضرع في األبقارعزل وتشخيص الفطريات من عينات الحليب المصابة المأ .الجبل على أهم العتر أآليللكحولي لنبات الفعالية الفطرية المثبطة للمستخلص ا أسيل إبراهيم العميد .جمهورية العراق – جامعة بغداد - آلية الطب البيطري - وحدة أألمراض المشترآة -مدرس مساعد * aseel-alam @ yahoo.com. المستخلص هذه الدراسة عزل وتشخيص الفطريات الموجودة في عينات الحليب المأخوذة من أألبقار استهدفت من العيادات 2009نيسان لسنة إلىالضرع، جمعت عينات الحليب خالل شهر شباط المصابة بالتهاب إصابات فطرية %) 80(عينة 80عينة حليب، أظهرت 100تم جمع. ية في منطقة أبوغريبلبيطرا ، )عينة17%(21.25وعزلت األعفان بنسبة ،)عينة 63% (78.75مختلفة ،عزلت الخمائر منها بنسبة ، تليها عزلة %25حيث عزلت بنسبة Candidaalbicans أغلب العزالت آانت لجنس Geotrichumcandidum 2نسبة ب Rhizopusثم عزلة ،% 0 spp. أما %.17.5بنسبة Candida spp. 1:اقي األجناس فكانت النسب آاألتيب 1 .2 ، جنس 5% Sacchromycescerevisiae 1 1 .2 5% ،Candida tropicalis 7.5% ، Cryptococcusneoformans 2.5%،Penicillium spp.2 .5% ، Rodotorulaspp Aspوأخيرا عزلة % 1.25. erg illu sterreu s و %1.25بنسبة ، 2 2(عينة 0 من % 70(لمستخلص الكحولي تم تحضير ا. لم تظهر إصابة فطرية%) 0 لنبات إآليل الجبل لدراسة تأثيره على نمو أهم الفطريات المعزولة من ) ألثيلياالكحول Caعينات الحليب والتي هي n did aa lbican s،Geot. candidum،Rhizopus spp.،Asp. terreus،Sacch . Cerevisiae .نتائج تأثير المستخلص بترآيز أظهرت ال Geot.candidum ،Sacch.cerevisiae ،Candidaمل على نمو الخمائر /ملغم200 albica ns 12،1، وآانت أقطار التثبيط آالتالي ملم بينما لم يظهر المستخلص أي 7،19 تثبيطي بقطرو Sacch.cerevisiaeعلى خميرة مل إال/ملغم100مثبط بترآيز تأثير 1 مل بالنسبة لعفن /ملغم10لنسبة لألعفان فكان الترآيز أألدنى للتثبيط هو اأما ب. ملم1 Asp. terreus الوسط الزرعي إلىعند إضافة المستخلصIn vitro وأظهرت Al – Ameed Diyala Agricultural Sciences Journal, 5( 2 ) 1 – 13 ,2013 13 20،40،8التراآيز مل أعلى نسبة تثبيط مقارنة باألطباق المعاملة بعقار /ملغم 0 Clotrimazole ترآيز أألدنى للتثبيط لعفن بينما آان الRhizopus spp مل /ملغم40 . يتبين مما . مل نسبة تثبيط قليلة مقارنة بالطبق المعامل بالعقار/ملغم80وأظهر الترآيز ألعفان من حليب أألبقار يدل على إلتهاب الضرع الفطري وقد اتقدم إن عزل الخمائر و ألثيلي لنبات ار المستخلص رع البكتيري، وقد أظهبالتهاب الض أإلصابةيحدث بعد ,Pineneألعفان وذلك إلحتواءه على مرآباتاجبل فعالية مثبطة لنمو الخمائر وإآليل ال Bornyl acetate, Camphor an d1,8 -Cin eole) -α (تؤثر في نموها. .ت إآليل الجبلعينات الحليب من أبقار مصابة بالتهاب الضرع، الفطريات، المستخلص الكحولي لنبا: الكلمات المفتاحية