KAFKAS ÜNİVERSİTESİ VETERİNER FAKÜLTESİ DERGİSİ

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2 ISSN: e-issn: KAFKAS ÜNİVERSİTESİ VETERİNER FAKÜLTESİ DERGİSİ THE JOURNAL OF THE FACULTY OF VETERINARY MEDICINE UNIVERSITY OF KAFKAS ÖZEL SAYI A: SUPPLEMENT A: MİKROBİYOLOJİ, TOKSİKOLOJİ ve HALK SAĞLIĞI MICROBIOLOGY, TOXICOLOGY & PUBLIC HEALTH (MART - NİSAN) (MARCH - APRIL) Cilt/Volume: 17 Sayı/Number: Supplement A Yıl/Year: 2011

3 This journal is indexed and abstracted by Thomson Reuters Services beginning with Volume 13 (1) 2007 in the followings: Science Citation Index Expanded (also known as SciSearch ) Journal Citation Reports/Science Edition This journal is also indexed and abstracted in: CAB Abstracts TÜRKİYE ATIF DİZİNİ ULAKBİM-TÜBİTAK EBSCO YAZIŞMA ADRESİ (Address for Correspondence) Kafkas Üniversitesi Veteriner Fakültesi Dergisi Editörlüğü Kars / Türkİye Phone: /1244 Fax: vetdergi@kafkas.edu.tr ELEKTRONİK BASKI (Electronic Edition) ISSN: ONLINE MAKALE GÖNDERME (Online Submission)

4 Bu dergi Kafkas Üniversitesi Veteriner Fakültesi tarafından iki ayda bir yayımlanır. This journal is published bi-monthly, by the Faculty of Veterinary Medicine, University of Kafkas. Kafkas Üniversitesi Veteriner Fakültesi Adına Sahibi (Owner) Prof.Dr. Hidayet Metin ERDOĞAN Dekan (Dean) EDİTÖR (Editor-in-Chief) Prof.Dr. İsa ÖZAYDIN YABANCI DİL EDİTÖRLERİ (English Editors) Doç.Dr. Ahmet ÜNVER Doç.Dr. Ş. Metin PANCARCI EDİTÖR YARDIMCILARI (Associate Editors) Prof.Dr. Mehmet ÇİTİL Doç.Dr. Özgür AKSOY Yrd.Doç.Dr. Oktay ÖZKAN Yrd.Doç.Dr. Duygu KAYA SEKRETERYA (Secretary) Fahri ALTUN DİZGİ (Composition) Özgür AKSOY BASKI (Print) ESER OFSET MATBAACILIK Tel: ERZURUM DANIŞMA KURULU (Advisory Board) Prof.Dr. Kemal AK İstanbul Üniversitesi Veteriner Fakültesi Prof.Dr. Belma ALABAY Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. Mustafa ALİŞARLI Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Prof.Dr. Feray ALKAN Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. Çiğdem ALTINSAAT Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. Metin BAYRAKTAR Fırat Üniversitesi Veteriner Fakültesi Prof.Dr. Burhan ÇETİNKAYA Fırat Üniversitesi Veteriner Fakültesi Prof.Dr. Hüdaverdi ERER Selçuk Üniversitesi Veteriner Fakültesi Prof.Dr. Gül ERGÜN Hacettepe Üniversitesi Fen Fakültesi Prof.Dr. Ayhan FİLAZİ Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. O. Sacit GÖRGÜL Uludağ Üniversitesi Veteriner Fakültesi Prof.Dr. Ekrem GÜREL Abant İzzet Baysal Üniversitesi Fen Edebiyat Fakültesi Prof.Dr. Tolga GÜVENÇ Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Prof.Dr. Ali İŞMEN Çanakkale Onsekiz Mart Üniversitesi Su Ürünleri Fakültesi Prof.Dr. Hakkı İZGÜR Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. Zafer KARAER Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. Arif KURTDEDE Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. Erdoğan KÜÇÜKÖNER Süleyman Demirel Üniversitesi Mühendislik Mimarlık Fakültesi Prof.Dr. Kamil ÖCAL Adnan Menderes Üniversitesi Veteriner Fakültesi Prof.Dr. Metin PETEK Uludağ Üniversitesi Veteriner Fakültesi Prof.Dr. Sevim ROLLAS Marmara Üniversitesi Eczacılık Fakültesi Prof.Dr. Engin SAKARYA Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. Berrin SALMANOĞLU Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. Nesrin SULU Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. Ş. Doğan TUNCER Ankara Üniversitesi Veteriner Fakültesi Doç.Dr. Cevdet UĞUZ Ayon Kocatepe Üniversitesi Veteriner Fakültesi Prof.Dr. Halis YERLİKAYA Fırat Üniversitesi Veteriner Fakültesi

5 Bu Sayının Hakem Listesi (alfabetik sıra) The Referees List of This Issue (in alphabetical order) AKAYLI Tülay AKSOY Abdurrahman AKSU Harun ALDEMİR Adnan ALTUĞ Nuri ALTUN Soner ARIMAN KARABULUT Huriye ATAKİŞİ Onur ATASEVER Mustafa AYDIN Levent BALTA Fikri ÇELEBİ Selahattin ÇIBIK Recep ÇİFTÇİOĞLU Gürhan EGEMEN Özdemir ELMAS Muammer GENÇ Oktay GÜL Yusuf HADİMLİ Hasan Hüseyin HASÖKSÜZ Mustafa KAMİLOĞLU Nadide Nabil KAR Sırrı KILIÇ Engin KIRPIK Mehmet Ali KUMLAY Ahmet Metin Mustafa ALPARSLAN NALBANTOĞLU Serpil NİZAMLIOĞU Mustafa ONUK Ertan Emek OTLU Salih ÖZKAN Oktay ÖZKUL Aykut ÖZTÜRK Savaş SARI Barış SARIMEHMETOĞLU Belgin SEL Tevhide SEZER Çiğdem SOLAK Kemal ŞAHİN Gönül ŞAHİN Mitat ŞAHİN Tekin ŞİMŞEK Sami TANYILDIZI Sadettin TAYAR Mustafa TURAÇLAR Nesrin TÜRK Necla ÜNVER Ahmet VATANSEVER Leyla VILCEK Stefan YARDIMCI Banu YARDIMCI Hakan YILDIRIM Yakup İstanbul Üniversitesi Su Ürünleri Fakültesi Su Ürünleri Yetiştiriciliği Bölümü Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Farmakoloji ve Toksikoloji Anabilim Dalı İstanbul Üniversitesi Veteriner Fakültesi Besin Hijyeni ve Teknolojisi Anabilim Dalı Kafkas Üniversitesi Fen Edebiyat Fakültesi Biyoloji Bölümü Mustafa Kemal Üniversitesi Veteriner Fakültesi İç Hastalıkları Anabilim Dalı Uludağ Üniversitesi Veteriner Fakültesi Mikrobiyoloji Anabilim Dalı Rize Üniversitesi Su Ürünler Fakültesi Su Ürünleri Yetiştiriciliği Bölümü Kafkas Üniversitesi Veteriner Fakültesi Biyokimya Anabilim Dalı Atatürk Üniversitesi Veteriner Fakültesi Besin Hijyeni ve Teknolojisi Anabilim Dalı Uludağ Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı Rize Üniversitesi Su Ürünler Fakültesi Su Ürünleri Yetiştiriciliği Bölümü Atatürk Üniversitesi Tıp Fakültesi Mikrobiyoloji Anabilim Dalı Uludağ Üniversitesi Veteriner Fakültesi Besin Hijyeni ve Teknolojisi Anabilim Dalı İstanbul Üniversitesi Veteriner Fakültesi Besin Hijyeni ve Teknolojisi Anabilim Dalı Ege Üniversitesi Su Ürünleri Fakültesi Temel Bilimler Bölümü Selçuk Üniversitesi Veteriner Fakültesi Farmakoloji ve Toksikoloji Anabilim Dalı Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Mikrobiyoloji Anabilim Dalı Fırat Üniversitesi Veteriner Fakültesi İç Hastalıkları Anabilim Dalı Selçuk Üniversitesi Veteriner Fakültesi Mikrobiyoloji Anabilim Dalı İstanbul Üniversitesi Veteriner Fakültesi Viroloji Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Fizyoloji Anabilim Dalı Namık Kemal Üniversitesi Fen Edebiyat Fakültesi Biyoloji Bölümü Kafkas Üniversitesi Veteriner Fakültesi Cerrahi Anabilim Dalı Kafkas Üniversitesi Fen-Edebiyat Fakültesi Biyoloji Bölümü Iğdır Üniversitesi Ziraat Fakültesi Tarla Bitkileri Bölümü Çanakkale Onsekiz Mart Üniversitesi Su Ürünleri Fakültesi Su Ürünleri Yetiştiriciliği Bölümü Ankara Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı Selçuk Üniversitesi Veteriner Fakültesi Besin Hijyeni ve Teknoloji Anabilim Dalı Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Su Ürünleri ve Hastalıkları Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Mikrobiyoloji Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Farmakoloji ve Toksikoloji Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Viroloji Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Cerrahi Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Biyokimya Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Besin Hijyeni ve Teknolojisi Anabilim Dalı Gazi Üniversitesi Gazi Eğitim Fakültesi Biyoloji Eğitimi Anabilim Dalı Hacettepe Üniversitesi Eczacılık Fakültesi Farmasötik Toksikoloji Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Mikrobiyoloji Anabilim Dalı Harran Üniversitesi Veteriner Fakültesi İç Hastalıkları Anabilim Dalı Fırat Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim Dalı Fırat Üniversitesi Veteriner Fakültesi Farmakoloji ve Toksikoloji Anabilim Dalı Uludağ Üniversitesi Veteriner Fakültesi Besin Hijyeni ve Teknolojisi Anabilim Dalı Selçuk Üniversitesi Sağlık Hizmetleri Meslek Yüksekokulu Bornova Veteriner Kontrol Araştırma Enstitüsü - İZMİR Kafkas Üniversitesi Veteriner Fakültesi Mikrobiyoloji Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Besin Hijyeni ve Teknolojisi Anabilim Dalı The University of Veterinary Medicine and Pharmacy in Košice, SLOVAKIA Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Su Ürünleri ve Hastalıkları Anabilim Dalı Ankara Üniversitesi Veteriner Fakültesi Mikrobiyoloji Anabilim Dalı Kafkas Üniversitesi Veteriner Fakültesi Viroloji Anabilim Dalı

6 İÇİNDEKİLER (CONTENTS) ARAŞTIRMA MAKALELERİ (Research Articles) Protective Effect of Caffeic Acid Phenethyl Ester Against Lead Acetate-induced Hepatotoxicity in Mice MUTLU N, ERSAN Y, NUR G, KOC E... Chemometric Classification of Some Elements in Wild and Farmed Bluefin Tuna (Thunnus thynnus L1758) SOGUT O, PERCIN F, KONYALIOGLU S... The Potential Risk in Epizootiology of Bacterial Zoonozis: Pigeon (Columba livia domestica) Feces ASKAR S, SAKARYA F, YILDIRIM M... Alpha-Amylase Production by Bacillus subtilis RSKK96 in Submerged Cultivation AKCAN N, UYAR F, GUVEN A... İzmit Körfezinden İzole Edilen Escherichia coli lerde R Plazmidlerine Bağlı Bulaşıcı Tipte Antibiyotik Direnç Özelliğinin Belirlenmesi VURAL HC, AKCIN A... Prevalence of Bacillus cereus in Rabbit Meat Consumed in Burdur - Turkey, Its Enterotoxin Producing Ability and Antibiotic Susceptibility KURSUN O, GUNER A, OZMEN G... The Prevention of the Development of Peritonitis with Different Disinfectants to Compare the Efficacy of Gastric Lavage at the NOTES SAKCAK I, AVSAR FM, YILMAZ EU, ATAS H, KILIC K, COSGUN E, MUMCUOGLU I, SEZER S, BOSTANOGLU S, ISGOREN A... Gastro-Intestinal Helminths Detected by Coprological Examination in Stray Dogs in the Erzurum Province -Turkey BALKAYA I, AVCIOGLU H... Balıklardan İzole Edilen Bakteriyel Patojenlere Karşı Bazı Bitkisel Uçucu Yağlarının Antibakteriyal Aktivitesi EKICI S, DILER O, DIDINEN BI, KUBILAY A... Presence of Staphylococcus aureus and Staphylococcal Enterotoxins in Different Foods KOLUMAN A, UNLU T, DIKICI A, TEZEL A, AKCELIK EN, BURKAN ZT... Bazı Salvia L. (Labiatae) Bitki Ekstraktlarının, Sivrisinek Culex pipiens L. (Diptera: Culicidae) e Karşı Larva Öldürücü Aktivitesi SEREF GUN S, CINBILGEL I, OZ E, CETIN H... Use of Chitosan in Turkish Sausage (Sucuk) Production and Effects on Quality GOKMEN M, GURBUZ U... The Alterations in the Hematological Parameters of Rainbow Trout, Oncorhynchus mykiss, Exposed to Cobalt Chloride ATAMANALP M, AKSAKAL E, KOCAMAN EM, UCAR A, SISMAN T, TURKEZ H... Evaluation of the Relationship of Blood Heavy Metal, Trace Element Levels and Antioxidative Metabolism in Cattle Which Are Living Near The Trunk Roads ARSLAN HH, SARIPINAR AKSU D, OZDEMIR S, YAVUZ O, OR ME, BARUTCU UB... Molecular and Serological Characterization of Pestivirus Infection Among Sheep in Kirikkale, Turkey AZKUR AK, GAZYAGCI S, ASLAN ME, UNAL N... Different Abundances of Human-Biting Ticks in Two Neighboring Provinces in Turkey GARGILI A, KAR S, YILMAZER N, ERGONUL O, VATANSEVER Z... Sayfa (Page) S1 S7 S13 S17 S23 S31 S37 S43 S47 S55 S61 S67 S73 S77 S83 S93

7 Sayfa (Page) Isolation of Citrobacters in Various Infections and Their Antimicrobial Sensitivity Rates KURTOGLU MG, OPUS A, OZDEMIR M, BAYSAL B... Ham Petrolün Suda Çözünebilen Kısımlarının Pelvicachromis pulcher (Boulenger, 1901) Cichlidae, Teleostei) Bağırsak ve Karaciğeri Üzerindeki Etkileri ONEN O, GUNDUZ O, ISISAG UCUNCU S... Determining Existance and Antibiotic Susceptibility Status of Listeria monocytogenes Isolated from Dairy Products, Serological and Moleculer Typing of the Isolates DUMEN E, ISSA G, IKIZ S, BAGCIGIL F, OZGUR Y, KAHRAMAN T, ERGIN S, YESIL O... Antibacterial and Antifungal Effects of Some Marine Algae ERTURK O, TAB B... Uçucu Yağ İçeren Yenilebilir Kitosan Filmlerinin Staphylococcus aureus ve Escherichia coli O157:H7 Üzerine Etkinlikleri TORLAK E, NIZAMLIOGLU M... Toxic Effects of Cadmium Sulphate on Tissue Histopathology and Serum Protein Expression in European Chub, Leuciscus cephalus (Linnaeus, 1758) YILMAZ M, ERSAN Y, KOC E, OZEN H, KARAMAN M... Bazı Bitki İnfüzyonları ve Hidrodistilatlarının Piliç Etlerinin Dekontaminasyonu ve Raf Ömrüne Etkisi AKSOY A, GUVEN A, GULMEZ M... Determination of Heavy Metal in Anchovy (Engraulis encrasicolus L 1758) and Whiting (Merlangius merlangus euxinus Nordman, 1840) Fish in The Middle Black Sea AYGUN SF, ABANOZ FG... Electrophoretical Comparison of Proteins of Mesobuthus eupeus and Mesobuthus gibbosus Scorpion Venoms OZKAN O, CIFTCI G, KARAER Z... Salmonella enterica serovar Typhimurium Suşunda MisL Ototransporter Proteininin İn Vitro Üretimi AKCELIK N, AKCELIK M... S99 S105 S111 S121 S125 S131 S137 S145 S153 S159 KISA BİLDİRİ (Short Communication ) Effectiveness of Different Disinfectants Used in the Food Production Facility on Selected Foodborne Pathogens MISIRLI F, AYDIN A... Investigation of Poly-β-Hydroxybutyrate (PHB) Production by Bacillus subtilis ATCC 6633 Under Different Conditions TAMDOGAN N, SIDAL U... S167 S173 OLGU SUNUMU (Case Report) İki Farklı Koyun ve Keçi Sürüsünde Brucella ve Campylobacter Ortak Enfeksiyonu BUYUK F, CELEBI O, SAHIN M, UNVER A, TAZEGUL E... S177

8 Kafkas Univ Vet Fak Derg 17 (Suppl A): S1-S5, 2011 DOI: /kvfd RESEARCH ARTICLE Protective Effect of Caffeic Acid Phenethyl Ester Against Lead Acetate-induced Hepatotoxicity in Mice Neslihan MUTLU * Yusuf ERSAN * Gökhan NUR ** Evren KOÇ *** * Kafkas University, Faculty of Science and Art, Department of Biology, TR Kars - TURKEY ** Kafkas University, Faculty of Veterinary Medicine, Department of Histology-Embriyology, TR Kars - TURKEY *** Kafkas University, Faculty of Veterinary Medicine, Department of Physiology, TR Kars - TURKEY Makale Kodu (Article Code): KVFD Summary The toxicity of lead in the liver seems to relate to the generation of reactive oxygen species (ROS). Caffeic acid phenethyl ester (CAPE), a flavonoid like compound, is one of the major components of honeybee propolis which has been demonstrated to have antioxidant, free radical scavenger. This study was designed to investigate the protective effects of the CAPE against lead acetateinduced hepatotoxicities in mice. A total of 25 mice were equally divided into 5 groups which were: 1 st control, 2 nd injected with intraperitoneally (IP) %1 ethanol. 3 rd injected with IP CAPE, 4 th injected with IP lead-acetate, 5 th injected with IP CAPE + lead acetate. Experimental animals were decapitated at the end of the study. Following systemic necropsies of the animals, tissue paraffin blocks were prepared and cut into 5 μm thick sections for hematoxylen-eosin staining. The samples were later monitored under a light microscope for histopathological changes. In conclusion, livers which were treated with 1% ethanol and CAPE were similar to control group. In the group which was treated with lead some degenerations and necrosis were observed. In the group which was treated with CAPE and lead, levels of necrosis and degenerations was observed in less degree. Keywords: Lead, CAPE, Histopathology, Liver Farede Kurşun Asetatla Oluşturulan Hepatotoksisiteye Karşı Kafeik Asit Fenetil Esterin Koruyucu Etkisi Özet Karaciğer de kurşun toksisitesinin reaktif oksijen türlerinin (ROS) üretimiyle ortaya çıktığı düşünülmektedir. Yapıca flavanoidlere benzeyen, bal arısı propolisinin aktif bir bileşeni olan kafeik asit fenetil ester (CAPE) in antioksidan ve serbest radikalleri yok edici özellikleri gösterilmiştir. Bu çalışma, farede kurşun asetatla oluşturulan hepatotoksisiteye karşı CAPE nin koruyucu etkisini araştırmak için dizayn edilmiştir. 1. grup kontrol, 2. grup intraperitonal (IP) yolla %1 etanol uygulanan grup, 3. grup IP olarak CAPE uygulanan grup, 4. grup IP olarak kurşun asetat uygulanan grup, 5. grup ise IP olarak CAPE + kurşun asetat uygulanan grup olmak üzere toplam 25 fare 5 gruba ayrılmıştır. Çalışma sonunda hayvanlar dekapite edildi. Hayvanların sistemik nekropsilerinin ardından elde edilen dokular parafin bloklara alınıp, hematoksilen-eozinle boyanmak üzere 5 μm lik kesitler alınmıştır. Örnekler histopatolojik değişiklikleri gözlemlemek için ışık mikroskobunda incelenmiştir. Sonuç olarak etanol ve CAPE uygulanan gruptaki hayvanlara ait karaciğer, kontrol grubu hayvanlarının karaciğer dokusuyla benzerdir. Kurşun uygulanan gruptaki hayvanların karaciğerlerinde bazı dejenerasyon ve nekrozlar gözlemlenmiştir. CAPE ve kurşunun birlikte uygulandığı grupta nekroz ve dejenerasyonların şiddetinin azaldığı görülmüştür. Anahtar sözcükler: Kurşun, CAPE, Histopatoloji, Karaciğer INTRODUCTION Lead is a toxic metal with no function in the body. As mines were taken out and started to be used on the earth, it spread into the biosphere and its amount in humans body significantly increased in parallel with industrialization 1. Upon widespread use of lead, both ecological balance has been unsettled and it has entered İletişim (Correspondence) /3108 nmutlu81@hotmail.com

9 S2 Protective Effect of Caffeic Acid... into the food chain resulting in vital health problems for humans 2. The most important lead contamination sources: lead added to petrol, lead-based paints, lead soldered conserved food cans, ceramic glasses and industrial pollutions 3. Lead may cause physiological, biochemical and behavioral malfunctions in the organism depending on amount 4. Main targets of the lead are hematopoetic system, neural system and renal system. Besides, reproduction system and immune system were recorded to have been damaged by metal 5. Also it is known to have undesired effects related with respiration and sight as well as hepatic system and reproduction 6. Caffeic acid phenethyl ester (CAPE), a flavonoid like compound, and is an active component of honey bee propolis 7. It has been used for alternative medicine in Middle Eastern countries for long years 8. It has been shown in many studies that CAPE has antioxidant 9, antiinflammatory 10, anti carcinogenic 11, antiviral 12, immunomodulator 13 anti-hepatoxic 14, neuroprotective 15 and antiatherosclerotic 16 effects. No hazardous effect of CAPE on normal cells has been reported so far 15. In a study comparing antioxidant effects of galangin, one of the propolis components, with CAPE, it was observed that both components clear reactive oxygen species. Moreover; CAPE was shown to clear oxygen radical and reactive oxygen species generated by xanthine oxidase system more effectively than galangin and decrease the malondialdehyde (MDA) level far better than galangin 17. In present study, lead acetate was given to mice to create hepatotoxicity and livers of the mice were investigated to observe protective effects by histopathological methods. As a consequence, the debate over whether or not CAPE will constitute a type of treatment for preventing potential damage to arise in case of exposure to lead will be clarified. MATERIAL and METHODS Animals and Treatment Twentyfive adult mice which are weeks old and weigh gram were used in this research. Mice were acclimated to laboratory conditions with regular temperature control ranging from 23±2 C and with balanced diet and water ad libitum. Mice were divided into the 5 groups and each group had 5 mice. Group 1 was injected with intraperitoneally (IP) 1 ml serum physiologic (0.9% NaCI), group 2 was injected with IP 1% ethanol (Carlo Erba,USA), group 3 was injected with IP 8.5 mg/kg CAPE ( 97% pure, Sigma Aldrich, USA), and group 4 was injected with 50 mg/kg lead in the form of lead acetate (Sigma Aldrich, USA) via IP means for 5 days and lead acetate was solved in 1 ml serum physiologic on a daily basis always before administration. Group 5 was injected with IP 8.5 mg/kg CAPE starting 3 days before the experiment, and injection of lead in the form of lead acetate with CAPE follow on for 5 days. CAPE solution was solved in 1% ethanol on a daily basis always before administration. Histopathologic Examinations After the last injection, liver tissues were dissected and received liver tissue was fixed in 10% formaldehyde solution for 48 h, and then paraffin blocks were prepared by means of routine histological methods. After that, cross sections with 5 m. thickness were taken from those blocks and stained by means of hematoxylen-eosin staining method, and finally histopathological changes were monitored under the light microscope. RESULTS Morphological Findings Macroscopic examination showed that bud-shaped oedema glands emerged in liver tissues of the animals given lead; liver was looked like faded. On the other hand, in the group administered CAPE together with lead, these formations were occured less and view of liver tissue was similar to the control group (Fig. 1). Microscopic Findings Light microscopic examinations demonstrated that liver tissues of the animals administered CAPE had a view similar to normal (Fig. 2) while liver tissues of those administered ethanol of 1% noted slight degeneration and necrosis causing from administering alcohol (Fig. 3). Severe degeneration, widespread necrosis areas and hydrophic and vacuolar degeneration were observed in the animals administered lead only (Fig. 4). Whereas these formations were occurred less in the animals administered CAPE together with lead (Fig. 5). DISCUSSION It was observed that severity of the degenerations was decreased in the animals administered lead together with CAPE in our study. Lead toxicity was studied by many scientist and its effects on various tissues and organs were determined 3,4,6, Slight oxidative stress on hepatocytes was seen in the liver of the animals administered lead 21. Daggett et al. 22 reported that lead application did not have effects on oxidative stress on rats kidney structure whereas it decreased glutathione (GSH) levels and increased malondialdehyde MDA levels in the liver tissue. Nakagawa 23 determined that GSH levels

10 S3 MUTLU, ERSAN NUR, KOÇ Fig 2. Microscopic view of the hepatocytes and sinusoidal structures in the liver tissue of the animals administered CAPE (Vc: Vena centralis), H-E x40 Şekil 2. CAPE uygulanan hayvanların karaciğer dokusunda hepatosit ve sinusoidal yapıların mikroskobik görünümü (Vc: Vena sentralis), H-E x40 Fig 3. Focal necrosis areas (N) and hydropic degeneration (arrows) in the liver tissue of the animals administered ethanol (Vc: Vena centralis), H-E x40 Şekil 3. Etanol uygulanan hayvanların karaciğer dokusunda hidropik dejenerasyon (oklar) ve fokal nekrozis bölgeleri (N) (Vc: Vena sentralis), H-E x40 Fig 1. Macroscopic view of the liver tissues obtained from the control and experiment groups. A- Control group, B- Lead + CAPE group, C- Lead group Şekil 1. Kontrol ve deneme gruplarından elde edilen karaciğer dokularının makroskobik görünüşü. A- Kontrol grubu, B- Kurşun + CAPE grubu, C- Kurşun grubu decreased depending on lead. In another study, Wang et al. 24 stated that administration to subcronical lead caused with decreases in hemoglobin, glutathione peroxidase (GSH-Px), superoxide dimutase (SOD) levels in the blood and GSH levels in the hepatocytes. It has been noted that

11 S4 Protective Effect of Caffeic Acid... Fig 4. Microscopic view of the liver tissue of the animals administered lead. Widespread necrosis areas (N), hydropic (white arrows) and vacuolar degeneration (black arrows) areas (Vc: Vena centralis), H-E x40 Şekil 4. Kurşun uygulanan hayvanların karaciğer dokusunun mikroskobik görünümü. Yaygın nekrozis bölgeleri (N), hidropik (beyaz oklar) ve vakuolar dejenerasyon (siyah oklar) bölgeleri (Vc: Vena sentralis), H-E x40 Fig 5. Microscopic view of the liver tissue of the animals administered lead together with CAPE. Focal necrosis areas (N), hydropic (white arrows) and vacuolar degeneration (black arrows) areas (Vc: Vena centralis), H-E x40 Şekil 5. CAPE ile birlikte kurşun uygulanan hayvanların karaciğer dokusunun mikroskobik görünümü. Fokal nekrosis (N) bölgeleri, hidropik (beyaz oklar) ve vakuolar dejenerasyon (siyah oklar) bölgeleri (Vc: Vena sentralis), H-E x40 nitric oxide (NO) level decreased due to the lead application 25. Similarly, the effects of the lead were determined histopathologically in animal models administered lead. Shalan et al. 6, reported that the expansion of sinusoidal structures, mononuclear cell infiltration and focal necrosis areas were occured depending on exposure to lead. Furthermore, El-Sokkary et al. 26, noted that application of 100 mg/kg lead caused necrosis and cytoplasmic vacuolization in the liver and degeneration and necrosis were seen in the kidney of the rats. Carmignani et al. 25, found out that intoxication rising upon administration of ppm lead in drinking water caused increases in hepatocytes activities in the liver. In our study, degeneration and necrosis areas in the liver were similar to abovementioned literature in the animals administered lead 6,25,26. Various mechanisms are involved in the process of ethanol-induced tissue impairment. Oxidative stress and its effects are among the most important. Most of the studies showed protective effects of the antioxidant agents in various models of ethanol induced injury 27. Protective function of CAPE on antioxidant system and other tissues was demonstrated in many studies. Cüre 7, studied on protective role of CAPE against oxidative stress rising upon excessive iron administration in rats, and reported that the increase in the level of MDA, by the application of iron decreased due to administration of CAPE. Lee et al. 14, revealed the increase of the MDA level and decrease of the GSH level in the oxidative stress occurred by carbon tetrachloride whereas CAPE application relieved the effect of such oxidative damage, and as a consequence of histopathological examinations, degenerations together with focal necrosis areas emerged in hepatocytes and hepatic cordones in the liver tissues, and CAPE application helped to relieve severity of these effects on the liver. In another study, vacuolization in molecular layer, neuroglial cell infiltration and picnotic cells were determined in prefrontal cortex sections obtained from rats exposed to cigarette smoke. On the other hand, structural changes caused by exposure to smoke were significantly decreased rats injected CAPE together with exposure to cigarette 28. Mollaoğlu et al. 29, identified the increase of the MDA level due to the cadmium effect and the decrease of NO level, but application of CAPE together with cadmium, NO level increased while MDA level decreased. In a similar study, Parlakpınar et al. 30, determined the increase of the MDA and NO levels while decreasing of SOD, CAT and GSH levels by the application of gentamisin (GEN), decreased MDA and NO levels while increased SOD, catalase (CAT) and GSH levels by the application of CAPE together with GEN. As a consequence of histopathological examination of kidneys revealed that tubular necrosis emerged apparently, but by the application of CAPE together with GEN severity of such necrosis decreased. In our study, it was found out that severity of degeneration decreased in liver tissues of the animals administered CAPE together

12 S5 MUTLU, ERSAN NUR, KOÇ with lead. This protective effect may possibly thought to take place by means of CAPE s inducing antioxidant system and thus decreasing oxidative damage. As a conclusion; lead which is widespread in the environment was toxic at 50 mg/kg dose, whereas CAPE had a preventive effect at the same dose of lead. In view of this data it may possibly was though that CAPE can be used for treatment purpose against potential hazardous effects that might rise in humans and other living creatures due to lead toxication. REFERENCES 1. Yüksel L: Kurşun ve çocuk. İst Çocuk Klin Derg, 31, , Yarsan E, Özdemir M, Turhan E: Sığırlarda Kurşunla Akut Zehirlenme Olgusu. Ankara Univ Vet Fak Derg, 43, , Grandjean P: Health Significance of Metals-Lead. 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13 Kafkas Univ Vet Fak Derg 17 (Suppl A): S7-S12, 2011 DOI: /kvfd RESEARCH ARTICLE Chemometric Classification of Some Elements in Wild and Farmed Bluefin Tuna (Thunnus thynnus L1758) Ozlem SOGUT * Fatih PERCIN ** Sibel KONYALIOGLU *** * Ege University, Faculty of Pharmacy, Analytical Chemistry Department, TR Bornova, Izmir - TURKEY ** Ege University, Faculty of Fisheries, TR Bornova, Izmir - TURKEY *** Ege University, Faculty of Pharmacy, Biochemistry Department, TR Bornova, Izmir - TURKEY Makale Kodu (Article Code): KVFD Summary Bluefin tuna Thnunnus thynnus 1758 (BFT) belongs to Scombridae family, is native species to subtropical regions of Atlantic Ocean, Mediterranean and Black Sea. The concentration some essential elements (Cu, Fe, Mg, Mn, Ni, Zn) and non-essential element (Pb) have been determined a total of 60 individuals of T. thynnus were collected: 30 captured in the wild and 30 sampled from a tuna farm using flame atomic absorption spectrometry. A preliminary chemometric study with the use of patttern recognition methods was carried out in order to characterise, classify and distinguish the different parts BFT. Multivariate chemometric techniques such as principle component analysis (PCA) and K nearest neighbour (KNN) are used to classify BFT to their type and origin on the basis of chemical data. Metal concentrations seem to be good candidates for classification system, as they are stable. This study clearly reported that PCA procedures appear useful tools for differentiation and classification of wild and farmed BFT using the profile of trace elements. Keywords: Trace metals, Thunnus thynnus, Atomic absorption spectrometry, Chemometric classification Doğa ve Çiftlik Orkinoslarında (Thunnus thynnus L1758) Tespit Edilen Bazı Elementlerin Kemometrik Sınıflandırması Özet Orkinos Thunnus thynnus 1758 (BFT) Scombridae familyasına ait olup, Atlantik Okyanusu, Ortadoğu ve Karadeniz in subtropik bölgelerinde yaşayan doğal türlerindendir. 30 adet doğadan yakalanan, 30 adet orkinos çiftiğinden alınan toplam 60 T. thynnus örneğindeki esansiyal elementlerin (Cu, Fe, Mg, Mn, Ni, Zn) ve esansiyal olmayan toksik elementin (Pb) konsantrasyonları alev atomik absorpsiyon spektrometresi kullanılarak tayin edilmiştir. Besleme orkinoslar, kafeslerden alınırken doğal olanları büyük balık ağlarıyla yakalanmışlardır. Orkinosların beslenme şekillerini ve farklı dokularının farklılandırılması, sınıflandırılması ve karakterize edilmesi için örnek tanımlama yöntemleri kullanılarak ön kemometrik çalışma yapılmıştır. Esas bileşen analizi (PCA), K en yakın komşular (KNN) gibi çok değişkenli kemometrik yöntemler, orkinosları türüne gore ve kimyasal bileşen temeline dayanarak sınıflandırmak için kullanılmıştır. Kararlı oldukları için metal konsantrasyonları sistemin sınıflandırılması için iyi bir aday olarak görülmektedir. Bu çalışma, PCA işlemlerinin doğada ve kafesde büyütülen orkinosların farklılıklarının ortaya konmasında ve sınıflandırılmasında yararlı bir yöntem olduğunu ortaya koymaktadır. Anahtar sözcükler: Eser elementler, Thunnus thynnus, Atomic absorpsiyon spektrometresi, Kemometrik sınıflandırma INTRODUCTION The bluefin tuna, Thunnus thynnus L. 1758, is a long- feed intensively with different nutrients and various aquatic lived and fast growing pelagic species with spawning species. Commonly, BFT are captured by purse seine in migratory behaviour 1. The bluefin tuna (BFT) migrate these period and it leads to the overfishing. Thus, BFT annually between the Black Sea and the Aegean and the should be listed as an endangered fish species in Atlantic Eastern Mediterranean. During the migratory session BFT Ocean, since İletişim (Correspondence) ozlem.sogut@ege.edu.tr

14 S8 Chemometric Classification of... The high commercial value and increasing demand make the bluefin tuna a suitable species for farming. Since the 2002s, a cage fattening industry has developed in Antalya Bay and Ildir Bay, located in Cesme-Izmir, Turkey. The quality of farmed tunas arises from several factors to do with farming and environment 2. Trace elements are required for normal life processes, and all animals including fish need these inorganic elements. Fish may derive these elements from diet and also from ambient water. Increased levels of trace elements are indicative of bioaccumulation of heavy metal toxins, mainly in liver tissues. Many fish species are threatened by such toxins and they are used to investigate the impact of environmental pollution 3-5. Copper (Cu) is an essential trace element for all organisms, including fish; it plays an important role in organism s metabolism and its concentration is well regulated. Cu is one of the most toxic metals to fish and has various effects, including increased liver enzyme activity, liver diseases 6. Iron (Fe) is another essential but potentially toxic element for animal organisms that is stored via a number of ways, including hemoglobin and myoglobin in erythrocytes, other proteins including ferritin and hemosiderin in serum and body tissues especially the liver. Iron is used for enzymatic reactions. Fe deficiency may leads to anemia, hemolytic problems and liver diseases 6. Magnesium (Mg) is essential like other macro elements in animal organisms. Mg serves as a cofactor of phosphohydrolases and phosphotransferases 7. Mg depletion (Hypomagnesemia) leads to weakness, tetany, erratic swimming and central nervous system disease, however, hypermagnesemia potential cardiac effects, renal disease. Manganese (Mn) is essential like other trace elements in animal organisms. Mn is necessary for the normal functioning of brain and proper lipid and carbohydrate metabolism. The absence of Mn in fish meal diet significantly influenced the mineral composition of common carp gonads 6. Nickel (Ni) can do no harm to an organism unless the organism absorbs the divalent nickel ion into its body or the nickel ion is bound strongly enough to a membrane (fish gills) so that the membrane cannot function properly. Ni deficiency in the liver is decreased concentrations of Zn in liver 8,9. Zinc (Zn) is essential element for aquatic life, for example, it occurs in the enzyme carbonic anhydrase, which catalyses the formation of carbonic acid from carbon dioxide in the blood. At high levels, Zn exert a direct toxic action 6. Although lead (Pb) has a high profile in human toxicology, it is of much lesser importance for aquatic life. The effects of Pb on central nervous system cause anemia, renal failure and increase of liver enzymes. Large predators, such as bluefin tuna, are at the top of aquatic food chains and hence they can accumulate large amounts of metals. In addition those pelagic organism, particularly tuna, are high-performance fish with very high metabolic rates and consequently the rate of food intake is elevated, a property which contribution notably to the accumulation of pollutants 10. The aim of this study was to develop a method to confirm the wild BFT as farmed BFT. In this work, we present the results of application of pattern recognition methods to key metals data in BFT to differentiate between wild and farmed ones. The basis of classification procedure was the metal accumulation of different parts of BFT samples. Metal concentration seems to be good candidates for classification system, as they are stable; although tunas are acknowledged to be top-level predators able to concentrate large quantities of metals and represent a dietary source for humans, to our knowledge only few papers have dealt with metal determination in the Mediterranean 2,9,11-14 and there is no classification paper on tunas. MATERIAL and METHODS Sampling Wild BFT were obtained using purse seiners from the bay of Antalya, Levantene Sea, during spring and early summer in Farmed BFT samples were taken from the cages of fish farm in Ildir Bay, Izmir, at the same time. The cages were conical, having 50 m surface diameter, 30 m depth and 30 m bottom diameter. The depth of sea was between 40 and 60 m. Physical parameters of sampling areas, like dissolved oxygen, temperature and salinity were measured by Oxyguard Handy Gamma (OxyGuard Int. A/S, Denmark). Fork length, total weight and gender of each BFT were determined. Wild samples were taken onto the deck approximately 15 min after capture. BFT were slaughtered and gutted and then liver samples were obtained and stored in CO 2 ice. Samples were transferred to the laboratory and washed with distilled water, dried in filter paper, homogenized, packed in polyethylene bags and stored at -80 C prior to analysis. Farmed samples were taken from the cages, transferred to the laboratory and prepared for analysis using the same method. Digestion Procedures Liver, heart and meat tissues (head and tail meat) with a wet weight of 0.5 g were dried for 48 h at 110 C. Samples were digested with a mixed solution of perchloric 70% and nitric 65% acids (3:7, v/v) and slowly heated 80 C until complete digestion. De-ionized water, from Milli-Q system (Millipore, Bedford, MA, USA) was used to prepare all aqueous solutions. The perchloric acid, nitric acid and standard solutions (1.000 mg/l) were supplied by Merck (Darmstadt, Germany). Apparatus A flame atomic absorption spectrophotometer (Varian SpectrAA-10 Plus model) was used for the analysis. The zinc, iron, nickel, manganese, copper and lead hollow

15 S9 SOGUT, PERCIN KONYALIOGLU cathode lamps were used according to the manufacturer s recommendations. Data Analysis Each sample was considered as an assembly of 7 variables represented by the chemical data. These variables called features formed a data vector which represented a BFT sample. Data vector belonging to the same group (wild or farmed) were analysed. Pattern recognition tools used in this work as follows: Autoscale This is the most widely used scaling technique. The procedure standardizes a variable m according to: Y mj ( X mj s X where Y mj is the value j for the variable m after scaling, x mj is the value j for the variable m before scaling. X m is the mean of the variable and sm is the standard deviation of the variable. The result is a variable with zero mean and a unit standard deviation. Standardization is useful in areas such as quantitative structure- properly relationships where many different pieces of information are measured on very different scales 15. Principal Component Analysis (PCA) This procedure was used mainly to achieve a reduction of dimensionality, i.e. to fit a j-dimentional subspace to the original p-variate (p> >j) space of the objects and pit allows a primary evaluation between-category similarity 14. K Nearest Neighbour (KNN) m m This method, based on the distance between objects in the p-space as its criterion, is used to classify an object in the category which contributes the greatest number of the K- nearest known objects. It is a non parametric method in as much as it does not formulate a hypothesis on the distribution of the variables used. Only the closest K objects are used to make any given classification. The importance of a given feature in taking the decisions is proportional to its contribution to the distance calculation. The inverse square of Euclidean distance was used in this work 14,15. Euclidean distance is the distance between two samples k and l is defined by: Y mj ( X mj s where there are j measurements and x ij is the j th measurement on sample i. Pattern recognition analysis were performed by means of statistical software packages MATLAB (Matlab 7.0) RESULTS X m m Ten water samples were analyzed for each site for each season, to check the water quality. Mean±SE values of dissolved oxygen, temperature and salinity parameters for Ildir Bay were 8.10±0.7 mgl -1, 15.3±1.5 C, 36.5±0.05 gl -1 in winter and 7.90±0.5 mgl -1, 15.7±2.2 C, 36.2±0.07 gl -1 in spring. Values for Antalya Bay were 7.85±0.2 mg L -1, 16.5±1.2 C, and 37.2±0.06 gl -1 in winter and 7.60±0.3 mgl -1, 17.0±1.7 C, 37.5±0.09 g L -1, respectively, in spring. A total of 60 individuals of T. thynnus were collected: 30 captured in the wild and 30 sampled from a tuna farm. In both wild and farmed BFT, 15 individuals were female and 15 were male. The fork length and weight of the 15 wild BFT were 1524±1.7 mm and 1530±1.4 mm, compared with 56.5±3.6 kg and 53.8 kg±3.2 kg for the farmed fish. The fork length and weight of female wild, female farmed, male wild and male farmed were 1540±1.9 mm and 58.6 kg±3.9 kg, 1551±2.1 mm and 55.5 kg±3.8 kg, 1508±1.2 mm and 54.4±2.7 kg and 1509±1.2 mm and 52.1 kg±2.4 kg, respectively. The results of the metals determined in wild and farmed and bluefin tuna samples are summarized in Table 1 and Table 2. The accumulation of the elements in the parts of the BFT was evaluated. PCA was used to provide a partial visualisation of data in a reduced-dimension plot. Examining score plot of samples in the space defined by the two first principle components in Fig. 1. A natural Table 1. Concentration of some trace metals in different parts Northern Bluefin Tuna (mg/kg wet weight) Tablo 1. Orkinosların farklı bölgelerindeki bazı eser element miktarları (mg/kg yaş ağırlık) Metal concentrations PARTS OF BFT Cu Fe Mg Zn Wild Farmed Wild Farmed Wild Farmed Wild Farmed Head 5.09± ± ± ± ± ± ± ±30.2 Tail 2.76± ± ± ± ± ± ± ±22.6 Heart 6.04± ± ± ± ± ± ± ±11.3 Liver 5.47± ± ± ± ± ± ± ±46.6 Mean±SE: Mean±Standart Error

16 S10 Chemometric Classification of... Table 2. Concentration of some trace metals in different parts Northern Bluefin Tuna (mg/kg wet weight) Tablo 2. Orkinosların farklı bölgelerindeki bazı eser element miktarları (mg/kg yaş ağırlık) Metal concentrations PARTS OF BFT Mn Ni Pb* Wild Farmed Wild Farmed Wild Farmed Head 1.12± ± ± ± ± ±0.26 Tail 0.96± ± ± ± ± ±0.30 Heart 1.23± ± ± ± ± ±0.36 Liver 1.29± ± ± ± ± ±0.23 Mean±SE: Mean±Standart Error * Pb is an non-essential toxic element separation between wild and farmed BFT was found. It is not only scores, however, that are of interest, but also sometimes the loadings. Exactly the same principles apply in that value of loadings at one PC can be plotted against that at the other PC. The results show in Fig. 2. Some loadings are in the middle of the loadings plots such as Cu, Fe, Mn and Zn. The KNN is a grouping method. Using nearest neighbour linkage and correlation coefficients for similarities, the dendrogram is presented in Fig. 3. It can be seen that accumulation of trace elements in both wild and farmed liver are very different from the others. The other objects appear to form two groups; wild and farmed. 6 Scores on PC# wliver cliver 0 ctail chead whead wtail Fig 1. Comparison of score plots according to the trace element levels of BFT -2 cheart wheart Şekil 1. Orkinoslardaki eser element düzeylerine göre skor noktalarının karşılaştırılması Scores on PC# 1 Loadings for PC# 1 versus PC# Fig 2. Comparison of loadings plots according to the trace element levels of BFT. Şekil 2. Orkinoslardaki eser element düzeylerine göre loading noktalarının karşılaştırılması Loadings for PC# Fe Mn Cu PbMg Zn -0.5 Ni Loadings for PC# 1

17 S11 SOGUT, PERCIN KONYALIOGLU 9 Dendrogram Using No Scaling and Distance on 3 PCs Fig 3. Dendrogram for trace elements in BFT by using PCA and KKN 1: Wild liver, 2: Wild head, 3: Wild tail, 4: Wild heart, 5: Farmed liver, 6: Farmed head, 7: Farmed tail, 8: Farmed heart Şekil 3. KKN ve PCA kullanılarak orkinoslara eser elementler için dendogram 1: Doğa karaciğer, 2: Doğa baş, 3: Doğa kuyruk, 4: Doğa kalp, 5: Kafes karaciğer, 6: Kafes baş, 7: Kafes kuyruk, 8: Kafes kalp Distance to K-Nearest Neighbor DISCUSSION Trace elements are essential in all living organisms and many are micronutrients in the diet of fish and mammals. Although the specific dietary requirements for trace elements (copper, iron, zinc, manganese, nickel, etc.) are largely unknown in fish, minor changes in the concentration of these elements have been shown to cause depressed health and growth states. Elevated metal concentrations in the marine environment are widespread and a potential health hazard to fish and mammals 17. The relationship between concentrations of trace elements in fish tissues, their harmful effects and greater information on the levels of these substances in fish and their predators are important because marine species form a significant component of many human diets. In this study, the accumulation of the some elements in different parts of both wild and farmed the BFT was evaluated. The levels obtained were similar to those found by other authors 2,9,11-14,18. The search for natural grouping in the samples is one of the main ways to study structure of the data. Principal component analysis (PCA) was used to provide a partial visualisation of data in a reduced-dimension plot. The principal components or eigenvectors are orthogonal and they are linear combination of the original variables 19. A natural separation between wild and farmed BFT was found by PCA (Fig. 1). In this factor space, wild BFT formed a group as like farmed ones. The standardisation puts all the variables on approximately the same scale. Hence the variables of low intensity assume equal significance to those of high intensity. If two scores plots provide comparable information, the less number of analyses are just as good as the full sets of tests. This can be of significant economical benefit. The loadings plots give some information about the analysis (Fig. 2). Some loadings are in the middle of the loadings plots such as Cu, Fe, Mn and Zn. These behave atypically and are probably not useful indicators of separation. Cu, Fe, Mn takes place as a group in Fig. 2. All of these are essential element for fish nutrition for nerve system and some enzymatic reactions. If excess amount of the elements are ingested and assimilated, toxicity may develop 6. Zn and the group of Cu, Fe, Mn are almost diametrically opposed, suggesting that they measure opposite properties. Most loadings are on an approximate circle. This is because standardisation is used, and suggested that we are probably correct in keeping only two components. Nickel shows discrimination but closer to Zn. It is suggested that the reason should be the relation between Ni level and Zn in liver. Ni deficiency in the liver is decreased concentrations of Zn in liver. The similarity of the new group from all other groups is given by the highest similarity of either of the original object to each other objects. KNN is a grouping method. The results of hierarchical clustering is presented in the form of dendrogram. The objects are organised in a row, according to their similarities. It can be seen that accumulation of trace elements in both wild and farmed liver are very different from the others in dendrogram obtained by using nearest neighbour linkage and correlation coefficients for (Fig. 3). The other objects appear to form two groups; wild and farmed. This study clearly shows that PCA procedures appear useful tools for differentiation and classification of wild and farmed BFT using the profile of trace elements. These findings might be a special relevance for farming in order to assure an adequate content nutrient of adapted. REFERENCES 1. Majkowsky J: Global fishery resources of tuna and tuna like species. FAO Fisheries, Techical paper, p. 483, 2007.

18 S12 Chemometric Classification of Vizzini S, Tramati C, Mazzola A: Comparison of stable isotope composition an inorganic and organic contaminant levels in wild and farmed bluefin tuna, Thunnus thynnus, in Mediterranean Sea. Chemosphere, 78, , Prudente M, Kim EY, Tanabe S, Tatsukawa R: Metal levels in some commercial fish species from Manila Bay the Philippines. Marine Pol Bull, 34, , Bustamante P, Gorrigue C, Breau L, Caurant F, Dabin W, Greaves J, Dodemont R: Trace elements in two odontocete species (Kogia breviceps and Globicephala macrorhnchus) stranded in New Caledonia (South Pasific). Environ Pol, 124, , Gümüş E, Erdoğan F: Effects of partial substation of fish meal with tuna liver meal on the fatty acid profile of Nile Tilapia Fry, Oreochromis niloticus. Kafkas Univ Vet Fak Derg, 16, S283-S290, Watanabe T, Kison V, Satoh S: Trace minerals in fish nutrition. Aquaculture, 151, , Hilton JW: The interaction of vitamins, minerals and diet composition in the diet fish. Aquaculture, 79, , Pane EF, Smith C, McGeer JC, Wood CM: Acute waterbone nickel toxicity in the mechanism of acute and chronic waterbone nickel toxicity in freshwater Cladoceran, Dalpinia magna. Environ Sci Technol, 37, , Ptashynski MD, Pedlar RM, Evans RE, Baron CL, Klaverkamp JF: Toxicity of dietary nickel in lake Whitefish (Coregonus cluppeafarmis). Aqua Toxicol, 58, , Storelli MM, Giacominelli-Stuffler R, Storelli A, Marcotrigiano GO: Accumulation of mercury, cadmium, lead and arsenic in swordfish and bluefin tuna from Mediterranean Sea: A comparative study. Mar Poll Bullet, 50, , Licata P, Trombetta D, Cristani M, Calo M, Celona A, Naccari F: Heavy metals in liver and muscle of bluefin tuna (Thunnus thynus) caught in the Strait of Messina (Sicily, Italy). Environ Monit Assess, 107, , Licata P, Trombetta D, Cristani M, Naccari C, Martina D, Calo M, Naccari F: 540 heavy metals in liver of Thunnus thynus from the coast of Southern Tyrrherian Sea. Toxicol Lett, 144, 145, Hellou J, Fancey LL, Payne JF: Concentrations of twenty-four elements in bluefin tuna, Thunnus Thynus from the Northwest Atlantic. Chemosphere 24, , Arslan Z, Secar DH: High resolution micromill sampling for analysis of fish otoliths by ICP-MS: Effect of sampling and specimen preparation on trace element fingerprints. Marine Environ Research, 66, , Brereton RG: Chemometrics, Data Analysis for the Laboratory and Chemical Plant. pp , John Wiley&Sons Ltd, England, Laterre MJ, Peno R, Pita C, Botana A, Garcia S, Herrero, C: Chemometric classification of honeys according to their type. II Metal content data. Food Chem, 66, , Jorgense D, Heisinger JF: The effect of selenium on the distribution of mercury in the organs of the black bulhead (Ictalurus melas). Comp Biochem Physiol Part C Comp Pharmacol, 87: , Nisbet C, Terzi G, Pilgir O, Sarac N: Determination of heavy metal levels in fish samples collected from the Middle Black Sea. Kafkas Univ Vet Fak Derg, 16 (1): , Sola-Larranaga C, Navarro- Blasco I: Chemometric analysis of minerals and trace elements in raw cow milk from the community of Navarra, Spain. Food Chem, 112, , 2009.

19 Kafkas Univ Vet Fak Derg 17 (Suppl A): S13-S16, 2011 DOI: /kvfd RESEARCH ARTICLE The Potential Risk in Epizootiology of Bacterial Zoonozis: Pigeon (Columba livia domestica) Feces Şinasi AŞKAR * Fatma SAKARYA * Murat YILDIRIM * * University of Kırıkkale, Faculty of Veterinary Medicine, Department of Microbiology, TR Kırıkkale - TURKEY Makale Kodu (Article Code): KVFD Summary The purposes of this study were to determine the profile of some Gram-negative bacteria in domestic pigeon intestinal microflora and isolated Enterococcus and E. coli strains to some antibiotics resistance were investigated by disc diffusion methods. For this purpose, cloacal swab samples were collected from a total of 215 pigeons from 8 different pigeon-houses, in Kirikkale, Turkey. A total of 180 isolates were obtained from 215 samples and these isolates were identified as 46% E. coli, 10% Enterobacter spp., 7% Shigella spp., 4.5% C. diversus, 3% K. ozoenae, %1.5 B. dispar, 1% P. alcalifaciens, 1% S. rubidae and 25% Enterococcus spp. It was determined that the isolates of E. coli were not resistant to enrofloxacine and gentamicine, while, they were found resistant to ampicilin-sulbactam, nalidixic acide and oxytetracyclin at the rates of 70%, 49% and 64% respectively. Moreover, 42.3% of the E. coli strains were identified as positive for β-lactamase activities. Keywords: Antibiogram, Escherichia coli, Enterococcus, Pigeons Özet Bakteriyel Zoonozların Epizootiyolojisinde Potansiyel Risk: Güvercin (Columba livia domestica) Dışkısı Bu çalışmada, hobi amacıyla yetiştirilen evcil güvercinlerin bağırsak florasındaki bazı gram negatif bakteri profilini belirlemek ve izole edilen E. coli ile enterokok suşlarının bazı antibiyotiklere direncini disk difüzyon metotu ile araştırmak amaçlandı. Bu amaçla Kırıkkale ve çevresindeki 8 güvercin kümesinden toplam 215 adet güvercinden kloakal svap örneği alındı. 215 numunede 180 izolat elde edildi ve bu izolatların %46 sı E. coli, %10 u Enterobacter spp., %7 i Shigella spp., %4.5 i C. diversus, %3 ü K. ozoenae, %1.5 ü B. dispar, %1 i P. alcalifaciens, %1 ü S. rubidae ve %25, Enterococcus spp. olarak identifiye edildi. E. coli izolatlarının enrofloksasin ve gentamisin karşı direnç taşımadığı, ampisilin-sulbaktam %70, nalidiksik asit %49, oksitetrasikline %64 dirençli olduğu tespit edildi. Ayrıca E. coli suşlarının %42.3 ünün, β-laktamaz aktivitesi yönünden pozitif olduğu belirlendi. Anahtar sözcükler: Antibiyogram, Escherichia coli, Enterokok, Güvercin INTRODUCTION The breeding of domestic pigeons (Columba livia domestica) are quite common in Turkey. Domestic pigeons pose the risk of contamination due to their close environment and contact with the other avian species, most importantly for humans because of carrying many bacteria. Lately, the increase in the number of pigeons leading to anxiety in terms of public health concerns 1. Pigeons are potential reservoir for humans that host many of patho- genic microorganisms with zoonotic features including Erysipelothrix insidiosa, Listeria monocytogenes, Pasteurella multocida, Yersinia spp., Campylobacter spp., Rickettsia rickettsii, Chlamydia, Salmonella spp., Enterobacter spp., Escherichia coli, Mycobacterium spp Pigeon breeding for domestic purposes have become quite popular and human contact with pigeon feces in public parks play an important role in the spread of infectious agents 7,8. Ishiguro et al. 9 were obtained 36 of E. coli isolates from İletişim (Correspondence) sinasia@gmail.com

20 S14 The Potential Risk in of pigeon cloacal swab samples taken from domestic pigeons, pigs, cattle and horses in their study. Tanaka et al. 10 conducted the study concerning the isolation and idenfication of Salmonella spp., Mycobacterium spp., E. coli O157 and Chlamydia spp. and they detected the presence of these bacteria in the samples of pigeon feces that were taken from 38 different public parks, located in 7 regions. Recently, the misusage of antibiotics have become widespread among the pigeon breeders, therefore, this situation may result in the emergence of antibiotic-resistant strains of microorganisms. Results reported by Kimpe et al. 11, revealed that the E. coli strains, isolated from pigeons showd resistance, to the antibiotics of ampicillin, gentamycin, kanamycin, trimethoprim/sulfamethoxasole, oxytetracycline, enrofloxacin, erythromycin. Silva et al. 8, obtained 184 of E. coli isolates from 100 fresh urban pigeon feces sampled in their study. This researchers indicated resistance profiles of 184 E. coli strains to Amikacin, Ampicillin, Ampicillin-sulbactam, Ceftazidime, Ceftriaxone, Gentamicin, Levofloxacin, Trimethoprimsulfametoxazole. Gülhan et al. 12 isolated 150 enterotoxigenic Echerichia coli (ETEC) strains from various animal feces samples. They reported, 7 enterotoxigenic E. coli isolates were detected in pigeon feces. The purposes of this study were to determine the profile of some Gram-negative bacteria in domestic pigeons intestinal microflora and isolated Enterococcus and E. coli strains to some antibiotics resistance were investigated by disc diffusion methods. MATERIAL and METHODS A total of 215 cloacal swab samples collected (197 tumbler, 14 ornamental, 4 carrier pigeons) from 8 pigeonhouses in Kirikkale and the surrounding region between September April The swab samples were inoculated directly onto agar Slanetz-Bartley (SB) agar and Eosine Methylene Blue (EMB) agar. The inoculated media were incubated at 37 C for h. The colonies that grew on EMB agar and resemble Enterobacteriaceae colony morphology were passaged into Tryptic Soy (TS) agar and incubated for 24 h. Then samples were identified by conventional test methods 13. The colonies that grew in SB agar and resemble Enterococ colony morphology, were subjected to catalase test and Gram staining. Catalase negative, PYR (L-pyrrolidonyl-beta-naphthylamide) positive, black colored in Bile Aesculin agar and grew in Brain- Heart Infusion (BHI) broth with 6.5% salt were considered as Enterococcus spp. This Enterococcus strains were tested for identification by arginine decarboxylation, motility test, pyruvate, mannitol, arabinose, raffinose and lactose fermentation test and pigmentation of grew colony in BHI agar 5,14. E. faecalis strains (ATCC ) were used as positive control for biochemical test. In order to determine the antibiotic resistance of E. coli strains, the discs (Oxoid) of enrofloxacin (5 μg), gentamicin (10 μg), oxytetracycline (30 μg), ampicillin/ sulbactam (10 μg/10 μg), nalidixic acid (30 μg) were used. To determine the antibiotic resistance of Enterococcus spp. the discs (Oxoid) of erythromycin (15 μg), enrofloxacin (5 μg), tetracycline (30 μg), quinupristin/dalfopristin (15 μg), penicillin G (10 U), teicoplanin (30 μg), streptomycin (5 μg), gentamicin (120 μg), vancomycin (30 μg) were used. The disc diffusion method in Mueller-Hinton (MH) agar was used for determining resistance to these antibiotics, according to Clinical and Laboratory Standards Institute -CLSI criteria 15. For detecting β-lactamase production in E. coli strains, β-lactamase (nitrocefin) identification sticks (Oxoid BR66A) were used. Test was performed according to the manufacturer s recommendations. The colonies that grew in MH agar applied onto sticks. Sticks were evaluated visually, for the color changes that observed in a humid environment at 5th, 15th and 30th. min intervals. RESULTS 180 isolates were obtained from 215 samples in this study. The isolates were identified as E. coli isolates, Enterococcus spp., Enterobacter spp., Shigella spp., C. diversus, K. ozoenae, B. dispar, P. alcalifaciens, S. rubidae (Table 1). Table 1. Bacteria isolated from cloacal swabs Tablo 1. Kloakal svaplardan izole edilen bakteriler Species of Microorganism Number of Isolate N (%) E. coli 83 (46) Enterococcus spp. 46 (25) Enterobacter spp. 18 (10) Shigella spp. 13 (7) C. diversus 8 (4.5) K. ozoenae 5 (3) B. dispar 3 (1.5) P. alcalifaciens 2 (1) S. rubidae 2 (1) According to the identification studies of isolated Enterococcus strains, the results of E. avium, E. dispar, E. raffinosus, E. faecalis asaccoharolytic variant were shown in Table 2. Enterococcus and E. coli strains to some antibiotics resistance patterns have been investigated by disc diffusion method. The resistance to antibiotics has been shown in Table 3 and Table of E. coli strains were found as positive (42.3%) in terms of β-lactamase activities.